Next Generation Sequencing For Cancer - NGS Cancer Testing
Next generation sequencing reads hundreds of cancer genes from one biopsy or a blood sample to find the mutations, fusions and markers that decide targeted therapy and immunotherapy. ESMO recommends it for advanced lung, prostate, bowel, bile duct, ovarian and breast cancer. Our team explains the report, flags inherited findings, and patients abroad can send tissue blocks or blood by courier without traveling.
About This Department
Medical Oncology
Two cancers that look the same under the microscope can be driven by different genes. NGS reads those genes and points to the drug that fits.
Next generation sequencing, or NGS, is a laboratory test that reads the DNA, and in good panels the RNA, of a tumor across dozens to hundreds of genes at once. It looks for the mutations, fusions and amplifications that drive the cancer, and for markers such as microsatellite instability and tumor mutational burden that predict response to immunotherapy. Tissue from a biopsy or operation already done works, and so does blood. The European Society for Medical Oncology now recommends tumor NGS in routine care for ten groups of advanced cancer, among them lung, prostate, bowel, bile duct, ovarian and breast cancer (Mosele and colleagues, Annals of Oncology, 2024). A result can open the door to a targeted drug, show that one will not work, reveal an inherited mutation that matters to the family, or qualify a patient for a clinical trial. This page explains how NGS works, which cancers need it, treatments that follow a gene regardless of where the cancer began, tissue and blood testing, the steps and timeline, how to read the report, and how patients abroad can send samples to Biruni Hospital in Istanbul.
10
Groups of advanced cancer for which ESMO recommends tumor NGS in routine practice as of 2024
75
Percent of patients with an NTRK fusion, across 17 tumor types, whose tumors shrank on larotrectinib
17.5
Percent of patients with advanced cancer found by paired tumor and blood sequencing to carry an inherited cancer gene
Free
Review of your pathology report and existing gene results by the medical oncology team
Older tests looked at one gene at a time, and each test used up part of a small biopsy. Next generation sequencing reads millions of short fragments of DNA in parallel and assembles them against the normal human sequence, so a single run covers many genes from one sample. A computer then lists every difference it finds, and a molecular pathologist decides which of them matter.
What NGS can find
Type of change
What it means
Example
Point mutation
One letter of the DNA code is changed
EGFR L858R in lung cancer, BRAF V600E
Fusion
Two genes break and join, making an abnormal protein
Osimertinib, alectinib, lorlatinib and other targeted tablets
Prostate cancer
BRCA1, BRCA2 and other repair genes
Olaparib and other PARP inhibitors
Bowel cancer
RAS, BRAF, MSI, HER2
Anti-EGFR antibodies, encorafenib, immunotherapy
Bile duct cancer
IDH1, FGFR2, BRAF, HER2, MSI
Ivosidenib, FGFR inhibitors
Ovarian cancer
BRCA1, BRCA2, homologous recombination deficiency
PARP inhibitors
Breast cancer
PIK3CA, ESR1, AKT1, PTEN, BRCA
Alpelisib, capivasertib, elacestrant
GIST, sarcoma, thyroid cancer, cancer of unknown primary
KIT, PDGFRA, RET, BRAF, NTRK and others
Imatinib, selpercatinib and others
ESMO recommends tumor NGS in advanced non-squamous lung, prostate, colorectal, bile duct and ovarian cancer, and since 2024 also in advanced breast cancer and in rare tumors such as gastrointestinal stromal tumors, sarcoma, thyroid cancer and cancer of unknown primary (Mosele and colleagues, 2024).
Treatments for any cancer type
Tumor-agnostic approvals
A few drugs are approved for a gene change wherever the cancer began, in the breast, the pancreas or a salivary gland. These alterations are rare in any single cancer, a few percent or less, so they are found only when someone looks. ESMO recommends NGS for these targets in any metastatic cancer where the matched drug is available.
Gene changes treated regardless of tumor type
Finding
Drug
Frequency
NTRK fusion
Larotrectinib, entrectinib, repotrectinib
Under 1 percent of common cancers, very common in some rare ones
MSI-high or mismatch repair deficiency
Pembrolizumab, dostarlimab
Around 4 percent of advanced cancers, more in womb and bowel
High tumor mutational burden
Pembrolizumab
Varies widely by cancer type
BRAF V600E
Dabrafenib with trametinib
Common in melanoma and thyroid, rare elsewhere
RET fusion
Selpercatinib
One to two percent of lung cancers, rare elsewhere
The overall response rate to larotrectinib was 75 percent in 55 patients with 17 unique TRK fusion-positive tumor types, ranging in age from 4 months to 76 years (Drilon and colleagues, 2018).
Why one test instead of many
Tissue runs out fast.
A lung biopsy taken with a needle may yield a few millimeters of tumor, and every single-gene test cuts slices from the same block. Testing EGFR, then ALK, then ROS1, then PD-L1 one after another can exhaust the sample before the last question is answered, and a second biopsy means another procedure and weeks of delay while the cancer grows. Time is lost too. Sequential testing also takes longer overall, because each result prompts the next request. One broad panel ordered at diagnosis answers every current question at once, and the laboratory rereads the stored data when a new drug is approved for a gene that was already on the panel. Studies in lung cancer have found that single-gene strategies miss a share of targetable changes that a broad panel catches, simply because the relevant gene was never tested.
The saving in time matters most in cancers that grow fast.
Tissue or blood
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Tissue first
The standard sample is tumor tissue fixed in formalin and set in paraffin, the same block the pathologist used for the diagnosis. The pathologist marks the area richest in tumor, and most panels need at least 20 percent tumor cells in the sample. Blocks up to several years old usually work, though fresher is better.
Tissue NGS and liquid biopsy
Feature
Tissue NGS
Liquid biopsy
Sample
Biopsy or surgical block
Two tubes of blood
Turnaround
About two weeks
About one week
Fusions and copy changes
Detected well
Detected less reliably
A negative result
Reliable when tumor content is adequate
Does not rule out a mutation if the tumor sheds little DNA
Best use
First choice at diagnosis
When tissue is scarce, a biopsy is risky, or to track resistance
When blood is used
Can a blood test replace a biopsy?
Not for the diagnosis, which still needs tissue. For gene testing, a liquid biopsy reads fragments of tumor DNA circulating in the blood, called ctDNA. A positive result is reliable and can start treatment, and in lung cancer many centers send blood and tissue at the same time to save days. A negative blood result is followed by tissue testing. Blood tests also track how a cancer changes on treatment, for example the EGFR resistance mutations that appear on osimertinib, without a new biopsy.
How the test works
1
From block to report
The oncologist requests the test and the pathologist selects the best block. Thin sections are cut, the tumor area is scraped off, and DNA and RNA are extracted and checked for quality. The genes on the panel are captured, copied and read on the sequencer. Software aligns the reads and lists the changes, and the molecular pathologist reviews each one against databases and the medical literature before signing the report.
Timeline
A typical timeline
Step
Time
Note
Block retrieval or shipping
One to five days
Longer when the block is held at another hospital
Extraction and sequencing
Five to seven days
A failed run is repeated if tissue allows
Analysis and report
Two to four days
Complex findings go to the molecular tumor board
Reading the report
Terms on an NGS report
Term
Meaning
Why it matters
Variant allele frequency
The share of DNA reads carrying the change
Around 50 percent can hint at an inherited mutation
Variant of uncertain significance
A change whose effect is unknown
Not acted on, may be reclassified later
Tumor mutational burden
Mutations per million DNA letters
10 or more may predict response to immunotherapy
MSI-high
Loss of the DNA spelling-check system
Strong response to immunotherapy
Actionable
Linked to an approved drug or a trial
The findings that change treatment
The alterations found, ranked by how much evidence links them to a treatment.
Matched drugs and trials.
Genome-wide markers, and a note if the sample was too small to assess them.
Levels of evidence
ESMO grades each finding on a scale called ESCAT. Tier I means a drug is proven to work for that alteration in that cancer and is ready for routine use. Lower tiers mean the evidence comes from another tumor type or from early studies, and the finding is a reason to look for a clinical trial rather than a prescription.
ESCAT tiers in plain words
Tier
Meaning
Action
I
Proven benefit in this cancer
Use the matched drug
II
Promising evidence awaiting confirmation
Consider, usually in a trial
III
Proven in another cancer type
Trial or off-label discussion
IV to X
Laboratory evidence only, or none
No action outside research
Inherited findings
A tumor test reads the cancer's DNA, but some of what it finds was present from birth in every cell. A BRCA2 mutation in a prostate cancer, for example, may be inherited, and brothers, sisters and children could carry it too.
Of 1,040 patients with advanced cancer, 182 (17.5 percent) had clinically actionable mutations conferring cancer susceptibility, and 101 (9.7 percent) would not have had these mutations detected using clinical guidelines (Mandelker and colleagues, JAMA, 2017).
Some panels sequence a blood sample alongside the tumor to separate the two. When only the tumor is tested and a suspicious finding appears, a blood test confirms whether it is inherited. Patients choose in advance whether they want to learn about inherited findings.
1
Genetic counseling
A confirmed inherited mutation leads to a genetic counseling appointment, which explains the risk to relatives, the screening or preventive surgery that may help them, and how family members can be tested in their own country.
When nothing is found
No actionable change
In many patients the report finds alterations that drive the cancer but have no approved drug yet. That result still guides care. It confirms that standard chemotherapy or immunotherapy is the right path and spares the patient a targeted drug that would not work. The data remain on file, and a new approval can make an old report newly useful.
What if the test fails?
Around one in ten tissue samples fail, mostly because too little tumor was present or the DNA was damaged by fixation. The team then tries another block, sends a liquid biopsy, or, if the answer would change treatment, arranges a new biopsy.
Tests NGS does not replace
Tests that are done by staining, alongside NGS
Test
Cancer
Why it is separate
Estrogen and progesterone receptors, HER2
Breast, stomach
Protein levels measured by staining the tissue
PD-L1
Lung, stomach, head and neck, cervix, others
Guides immunotherapy, measured by staining
Mismatch repair proteins
Bowel, womb, others
A quick staining screen that complements MSI by NGS
HER2-low and ultralow
Breast
Staining grades that decide trastuzumab deruxtecan
NGS reads genes. Staining measures proteins. Both are needed to plan modern cancer treatment.
The pathologist runs the staining tests on the same block, and the oncologist reviews both results together.
1
The molecular tumor board
Unusual or complex results are discussed by oncologists, pathologists and geneticists together, who match them to drugs and trials and write a single recommendation for the treating doctor.
Repeating the test
A cancer changes under treatment.
When a targeted drug stops working, a new test finds the reason, a second mutation in the same gene or a switch to another pathway, and points to the next drug. A liquid biopsy suits this well. A new tissue biopsy of a growing deposit gives the fullest answer when safe.
Timing matters as well. Testing at diagnosis sets the first treatment. Testing again at progression sets the second, and in lung cancer with an EGFR mutation, looking for the resistance change called C797S or a new MET amplification has become part of routine follow-up at centers that treat patients over many years of targeted therapy.
For patients abroad
Sending samples without traveling
NGS is one of the few services that does not require a trip. Your hospital sends the paraffin block, or unstained slides cut from it, by courier together with the pathology report, and a blood sample for liquid biopsy can be drawn locally and shipped in special tubes that keep the DNA stable for several days. The report goes to you and your oncologist. A video consultation with our oncologist then explains what it means for your treatment, what to request from your doctors at home and whether a clinical trial is worth considering.
1
Length of stay if you come in person
About two weeks. That covers consultation, a biopsy if needed and the test itself. Patients are fit to fly the day after most needle biopsies, and results can be discussed by video once you are back home.
One coordinator from the international patients office handles arrangements from the first message through discharge and answers the same WhatsApp number once you are back home. The office works in English, Arabic, French, Russian, Serbian, Romanian and Spanish and books interpreters for other languages, and it arranges the visa invitation letter, airport transfers, daily transport and accommodation near the hospital. Halal, vegetarian and diabetic meals, a prayer room on site, and a female physician on request wherever the rota allows.
Cost
What a quote for NGS covers
Item
Included
Priced separately
The test
Sequencing of the agreed panel, DNA and RNA where specified
A second run on another block, liquid biopsy
Interpretation
Molecular pathologist report, tumor board review of complex findings
Genetic counseling and blood confirmation of inherited findings
Consultation
Explanation of the result by an oncologist
Treatment that follows
The price depends on the size of the panel, whether RNA is read and whether blood is sequenced alongside the tumor.
1
Before you compare quotes
Ask how many genes the panel reads, whether it detects fusions through RNA, whether it reports MSI and tumor mutational burden, and how long the result takes.
A cheaper small panel that misses the relevant gene costs more in the end.
Shipping of blocks and blood tubes is arranged by the coordinator and quoted with the test.
References
Mosele MF, Westphalen CB, Stenzinger A, et al. Recommendations for the use of next-generation sequencing (NGS) for patients with advanced cancer in 2024. A report from the ESMO Precision Medicine Working Group. Ann Oncol. 2024;35(7):588-606.
Drilon A, Laetsch TW, Kummar S, et al. Efficacy of larotrectinib in TRK fusion-positive cancers in adults and children. N Engl J Med. 2018;378(8):731-739.
Mandelker D, Zhang L, Kemel Y, et al. Mutation detection in patients with advanced cancer by universal sequencing of cancer-related genes in tumor and normal DNA vs guideline-based germline testing. JAMA. 2017;318(9):825-835.
Zehir A, Benayed R, Shah RH, et al. Mutational landscape of metastatic cancer revealed from prospective clinical sequencing of 10,000 patients. Nat Med. 2017;23(6):703-713.
Editor's note
Written by the Biruni Hospital medical editorial team. Reviewed by Prof. Dr. Metin GÜDEN, Radiation Oncology.
