← Back
Diagnosis and tests

Molecular tests: PCR and NGS

Some changes in leukemia are so small that no microscope can show them — they may be an error in just a few “letters” of DNA. Molecular tests work at exactly this level. This topic explains what PCR and next-generation sequencing (NGS) are, which questions they answer and what their results mean for choosing treatment and tracking the response to it.

Last updated: ~6 min read
A translucent glowing DNA helix stretching across a dark navy background; along one section its rungs are highlighted in warm golden light, as if being read.
Figure 1.Concept: molecular tests read the “letters” of DNA.

01Short answer

Molecular tests read the DNA or RNA of leukemia cells directly. PCR (polymerase chain reaction) copies a small target piece of DNA over and over — up to billions of copies — which makes it very sensitive and able to find even a very small number of leukemia cells. Quantitative PCR measures how much of a change is present — for example, in CML the BCR::ABL1 level is tracked throughout treatment on an International Scale. NGS (next-generation sequencing) reads dozens or even hundreds of genes at once and helps identify mutations such as FLT3, NPM1, IDH1, IDH2 and TP53 in AML, and TP53 and IGHV status in CLL. These results refine the diagnosis, define the risk group and open the way to targeted drugs. After treatment, molecular tests are used to track measurable residual disease (MRD). The changes in leukemia cells are usually not inherited, but in rare cases a test may point to an inherited predisposition — in that case an additional test and genetic counselling are offered.

02Simple explanation

DNA is a long set of instructions written in four “letters”. Sometimes only a few letters change in a leukemia cell, yet this can completely change how the cell behaves. Molecular tests are designed to find such small errors.

PCR is like a “molecular photocopier”: it copies a small target piece of DNA again and again so that it can be seen and measured. That is why PCR is so sensitive — it can find the odd leukemia cell hidden among thousands of normal ones.

NGS is like reading a whole chapter of a book at once instead of a single page: it checks many genes at the same time. This matters especially in leukemias such as AML, where several different mutations can guide treatment.

The results answer three questions for the doctor: what exactly is the type of leukemia, how high is the risk and which medicines may be especially helpful. Some mutations have dedicated targeted drugs.

Molecular tests keep working during treatment too: they show how deeply treatment has worked. In CML, for example, a regular blood test tracks how the BCR::ABL1 level falls. Always discuss the results with your hematologist.

03Scientific explanation

PCR and RT-PCR: according to the NHGRI, PCR is a fast technique for “amplifying” small segments of DNA; over 30–40 cycles it produces more than a billion exact copies of the original segment. According to the NCI, RT-PCR first converts RNA into matching DNA and shows whether a particular gene is active; fusion genes (BCR::ABL1, PML::RARA) are often measured this way. The ACS notes that PCR finds small changes that cannot be seen under the microscope and is good at detecting the small numbers of leukemia cells left after treatment.

NGS reads many stretches of genes in parallel (NHGRI). According to the ACS, AML cells are usually tested for changes in genes such as FLT3, IDH1, IDH2, NPM1 and KMT2A, and people with these changes are more likely to be helped by certain targeted drugs. In CLL, a TP53 mutation is linked with a more guarded outlook and affects the choice of treatment, while IGHV mutation status helps predict the course of the disease (ACS). In hairy cell leukemia, according to the NCI, the BRAF V600E change is a disease-defining genetic feature that aids diagnosis.

What might the report show? The gene and the change (for example, “NPM1 type A” or “FLT3-ITD”), the variant allele frequency (VAF — the percentage of DNA in the sample carrying the change) and how significant the change is: pathogenic, likely pathogenic or a “variant of uncertain significance” (VUS). ELN 2022 places an NPM1 mutation (without FLT3-ITD) in AML in the favourable group and a TP53 mutation in the adverse group. The VAF can sometimes also hint that a mutation might be inherited.

Monitoring and inherited questions: under the 2020 ELN recommendations for CML, BCR::ABL1 is measured on the International Scale (IS); ≤10% at 3 months, ≤1% at 6 months and ≤0.1% (major molecular response) at 12 months are considered optimal. In AML, the 2021 ELN consensus recommends quantitative PCR for MRD in NPM1-mutated leukemia. The NCI notes that biomarker testing of a cancer is different from testing for inherited mutations but can sometimes reveal a change that might be inherited; in that case a separate confirmatory test and genetic counselling are recommended.

04What happens in the body?

Sample and timing

Molecular tests are usually run on bone marrow aspirate or blood. Some tests that give urgent information (for example, FLT3 or PML::RARA) are done as quickly as possible, because they can change the choice of treatment. Broad NGS panels can take longer. Ask your doctor which result will come back when.

PCR: making a tiny trace bigger

In every cycle, PCR doubles the number of copies of the target DNA. Within a few hours an invisible trace becomes a measurable signal. Quantitative PCR works out how much of the change is present from how quickly that signal appears — this is the basis of MRD and CML monitoring.

Small glowing DNA fragments on a dark navy background that double in number from left to right: one, two, four, eight — ending in a dense, bright cloud on the right.
Figure 2.PCR copies a small target piece of DNA again and again.

NGS: reading many genes at once

In NGS the DNA is broken into small fragments, millions of fragments are read in parallel and a computer puts them back together. The result is a whole “genetic profile” of the leukemia from a single test. This profile can change over time, so the test may be repeated at relapse.

Many thin glowing strands of DNA running in parallel across a dark navy background; a few of them carry small points of violet light.
Figure 3.NGS reads many genes at once and produces a genetic profile of the leukemia.

From result to treatment

Molecular findings can open the way to targeted drugs: FLT3 inhibitors, IDH1 and IDH2 inhibitors, menin inhibitors and, in CML, tyrosine kinase inhibitors. But the choice of drug rests on the whole picture, not on one result. Discuss with your hematologist which options are realistic for you.

05Molecular tests step by step

Four steps — from the sample to the treatment plan.

DNA/RNA — Extracted from marrow or blood cells.1DNA/RNAExtracted from marrow or blood cells.
PCR — A specific change is copied and measured.2PCRA specific change is copied and measured.
NGS — Many genes are read at once.3NGSMany genes are read at once.
Interpretation — Risk group and targeted treatment options.4InterpretationRisk group and targeted treatment options.
Genetic profileFLT3, NPM1, IDH, TP53 and more.
Targeted drugsChoosing treatment to match the mutation.
MRD and monitoringFor example, the BCR::ABL1 level in CML.

06Commonly tested molecular markers (simplified)

MarkerWhere?Why does it matter?
BCR::ABL1 (quantitative PCR)CML, Ph-positive ALLDiagnosis and tracking response to treatment (IS)
PML::RARAAPLConfirming the diagnosis and MRD
FLT3 (ITD, TKD)AMLFLT3 inhibitors; risk assessment
NPM1AMLRisk group; MRD by PCR
IDH1, IDH2AMLIDH inhibitors
TP53AML, CLLAdverse outlook; choice of treatment
IGHV mutation statusCLLPredicting the course of the disease
BRAF V600EHairy cell leukemiaSupports the diagnosis

* This table is a simplified summary; the list of tests varies by clinic, type of leukemia and treatment plan. Discuss what your results mean with your hematologist.

07Key terms

PCR
Polymerase chain reaction — a test that copies a small piece of DNA many times.
RT-PCR
A type of PCR in which RNA is first converted into DNA and then measured.
Quantitative PCR
PCR that measures how much of a change is present.
NGS
Next-generation sequencing — a test that reads many genes at once.
Mutation
A change in the DNA sequence.
VAF
Variant allele frequency — the percentage of DNA carrying a change.
VUS
A genetic variant whose significance is not yet known.
International Scale (IS)
A standard for comparing BCR::ABL1 results in CML.
Somatic change
A change acquired during life in some cells only; not inherited.
Genetic counselling
A conversation with a specialist about inherited risk, with support in decisions.

08Key points

  • Molecular tests find small changes in DNA and RNA.
  • PCR is very sensitive and is used for MRD.
  • Quantitative PCR tracks the BCR::ABL1 level in CML.
  • NGS reads many genes at once.
  • Results define the risk group and targeted drug options.
  • Some urgent results can change the choice of treatment.
  • Changes in leukemia are usually not inherited.
  • If an inherited change is suspected, a separate test and counselling are offered.

09Frequently asked questions

What is the difference between PCR and NGS?

PCR searches for and measures a specific, already known change with great sensitivity. NGS reads many genes at once and can also find unexpected mutations. They are often used together.

My report says “VUS”. Should I worry?

VUS means a variant whose significance is not yet known. Treatment decisions are usually not based on such variants. Ask your hematologist or a genetic counsellor what it means for you.

Will my mutation be passed on to my children?

Most mutations in leukemia cells are acquired during life and are not inherited. In rare cases a test points to a change that might be inherited; your doctor may then offer a separate confirmatory test and genetic counselling.

Why is treatment started before all the results are back?

In acute leukemia a delay can be dangerous. Doctors sometimes start treatment and then refine the plan as molecular results arrive — for example, by adding a targeted drug.

How often is BCR::ABL1 checked in CML?

Usually every few months at the start of treatment, then less often once the response is stable. Your hematologist will set the exact schedule. Where possible, it is important to use the same laboratory, one that reports on the IS standard.

Is the test repeated at relapse?

Yes, often. The genetic profile of a leukemia can change over time, and new mutations can open new treatment options. That is why molecular tests are usually repeated at relapse.

10Questions for your doctor

Take these questions to your appointment. The ones you save are collected on the “My questions” page and stay on this device only.

  1. Which changes did the PCR or NGS tests find?

  2. Which results affect the choice of treatment?

  3. Will these tests be used for MRD monitoring?

  4. If tests are sent abroad, how long will the results take?

My questions

12Sources

  1. National Cancer Institute (NCI). NCI Dictionary of Cancer Terms: polymerase chain reaction
  2. National Cancer Institute (NCI). NCI Dictionary of Cancer Terms: next-generation sequencing
  3. National Human Genome Research Institute (NHGRI). Polymerase Chain Reaction (PCR) Fact Sheet
  4. National Human Genome Research Institute (NHGRI). DNA Sequencing Fact Sheet
  5. National Cancer Institute (NCI). Biomarker Testing for Cancer Treatment
  6. National Cancer Institute (NCI). Genetic Testing Fact Sheet
  7. National Cancer Institute (NCI). Adult Acute Myeloid Leukemia Treatment (PDQ®) — Patient Version
  8. National Cancer Institute (NCI). Hairy Cell Leukemia Treatment (PDQ®) — Health Professional Version
  9. American Cancer Society. Tests for Acute Myeloid Leukemia (AML)
  10. American Cancer Society. Tests for CML
  11. American Cancer Society. Tests for CLL
  12. MedlinePlus (U.S. National Library of Medicine). BCR-ABL1 Genetic Test
  13. Blood (American Society of Hematology), 2022. Diagnosis and management of AML in adults: 2022 recommendations from an international expert panel on behalf of the ELN
  14. Blood (American Society of Hematology), 2021. 2021 Update on MRD in acute myeloid leukemia: a consensus document from the European LeukemiaNet MRD Working Party
  15. Leukemia, 2020. European LeukemiaNet 2020 recommendations for treating chronic myeloid leukemia
Medical disclaimerThis information is for educational purposes only and does not replace medical advice.
Last updated: ← Back to the list

Open the interactive site →