CME
Physicians: Maximum of 0.50 AMA PRA Category 1 Credit™
Released: July 10, 2025
Expiration: January 09, 2026
FLT3 Is the Most Commonly Mutated Gene in AML
Harry P. Erba, MD, PhD
An estimated 22,010 new cases of AML will be diagnosed and 11,090 AML-related deaths will occur in 2025 in the United States.8 AML is a very aggressive leukemia and there is a great unmet need to improve patient outcomes.
The cytogenetic and mutational underpinnings of AML are important prognostic indicators. Most patients have at least 1 driver mutation, with the 3 most common being in FLT3, NPM1, and DNMT3A.9 These mutations fall into different classes. Receptor tyrosine kinase mutations affect genes such as KIT and FLT3. Other groups of mutations affect spliceosomes, chromatin modifiers, and transcription factors such as RUNX1.
The General Pattern of AML Pathogenesis
Mark J. Levis, MD, PhD
Although numerous mutations are observed in AML, the typical patient with AML will only have 2-5 of these mutations and they tend to occur in a pattern. This is a simplistic model, but AML typically begins with a founder mutation that may be in an epigenetic regulator gene such as DNMT3A.10-13 Patients can have a mutation for years without symptoms, which is known as clonal hematopoiesis of indeterminate potential.
Then a cooperating mutation may develop. The cooperating mutations include transcription factors and regulatory genes such as RUNX1, CEBP-α, and NPM1. Signaling pathway mutations in FLT3, RAS, or PTPN11 are almost always the last hit in rendering a stem cell population into a clone that emerges as AML.12
Characteristics of FLT3 Mutations in AML
Mark J. Levis, MD, PhD
FLT3 is a cytokine receptor that signals predominantly through the RAS pathway. There are 2 major categories of FLT3 mutations known as FLT3-TKD and FLT3-ITD. Mutations in the tyrosine kinase domain of the activation loop activate FLT3, which are the FLT3-TKD mutations.1,14 The most common affected FLT3 residues are D835 or D836.
The coding sequence of FLT3 is conducive to the development of tandem duplications within the juxtamembrane domain, and these are known as FLT3-ITD mutations. FLT3-ITD mutations constitutively activate the kinase and the presence of FLT3-ITD mutations has a negative effect on AML prognosis.
FLT3-TKD mutations occur in 5% to 10% of AML cases, and are less common than FLT3-ITD mutations, which occur in approximately 25% of cases.1
FDA-Approved FLT3 Inhibitors for AML
Mark J. Levis, MD, PhD
Knowing whether a patient has an FLT3-ITD or FLT3-TKD mutation is not only important prognostically, but it also potentially affects therapy recommendations.15,16 FLT3-ITD mutations are inhibited by both type I and type II FLT3 inhibitors. Type II inhibitors, such as quizartinib, are generally more selective and are typically less effective against the FLT3-TKD mutations.
Type I inhibitors such as midostaurin and gilteritinib bind to the FLT3 active site regardless of the conformation of the activation loop. By contrast, type II inhibitors bind to a hydrophobic groove extending from the active site, and they can only bind to the inactive conformation.1
When a patient has an FLT3-TKD mutation, the receptor toggles between active and inactive conformations, making it difficult for type II inhibitors to bind effectively. This is one important reason why you cannot group all FLT3 mutations together; you need to know which type of mutation a patient has.
AML Survival by FLT3 Mutation Type
Harry P. Erba, MD, PhD
FLT3-TKD mutations have an unclear impact on prognosis, which is likely driven more by comutations in genes such as NPM1, chromatin modifiers, or spliceosomes. By contrast, in the era before FLT3 inhibitors, it was clear that there were patients who presented with normal karyotype AML, which accounts for approximately 50% of AML, and a very high white cell count. This patient population is typically enriched for FLT3-ITD mutations and has an inferior outcome compared with patients who do not have FLT3-ITD mutations.
These datasets from Germany and the United Kingdom show that patients with an FLT3-TKD mutation have similar overall survival to those without an FLT3 mutation.2,3 However, patients with FLT3-ITD mutations had worse outcomes, such that at 10 years, twice as many patients with an FLT3-TKD mutation were alive compared with those with an FLT3-ITD mutation.
FLT3-ITD Affects Outcomes After Allogeneic HSCT
The impact of the FLT3-ITD mutation on outcomes is also evident in patients receiving allogeneic transplants. There are 2 data sets that support this. A retrospective analysis of 206 adults with FLT3-ITD–mutated AML who underwent myeloablative allogeneic transplant showed a much higher likelihood of relapse in those with FLT3-ITD mutation compared with those with an FLT3-TKD mutation or those without an FLT3 mutation (HR: 3.4).4 Similarly, a retrospective analysis of 262 patients with AML who underwent allogeneic hematopoietic stem cell transplantation found a much higher 3-year relapse risk in a multivariate analysis (HR: 3.63; 95% CI: 2.13-6.19; P <.001).17
Prognostic Impact of FLT3-ITD Allelic Ratio
In the 1990s and 2000s, multiple studies found that the amount of the FLT3-ITD mutation, as measured by allelic ratio, affected prognosis.18 A high allelic ratio not only affected event-free and overall survival but also complete remission rate.
AML Risk by FLT3 Status in 2017 and 2022 ELN Risk Classifications
In the ELN 2017 risk stratification (before we started using FLT3 inhibitors with intensive chemotherapy) patients with FLT3-ITD mutations with a high allelic ratio were classified as having adverse risk disease in the presence of wild-type NPM1.19 A lower allelic ratio with wild-type NPM1 or a high ratio with an associated NPM1 mutation was considered intermediate risk. FLT3 inhibitors are now improving outcomes of patients with FLT3-ITD–mutated disease. The 2022 ELN risk classification now considers FLT3-ITD mutations, regardless of the allelic ratio or NPM1 mutation status, as intermediate risk.19
Mark J. Levis, MD, PhD
Of note, the 2022 ELN classification is predicated on the assumption that FLT3 inhibitors are being used early and often.
Real World Data: Incidence of FLT3-ITD Mutations in AML Subtypes
These real-world data from our institution show that FLT3-ITD mutations are found in all subtypes of AML and in some patients with myelodysplastic syndromes. Similarly, FLT3-TKD mutations are also found across the various AML subtypes. Thus FLT3-mutant AML does not have its own WHO classification. It also demonstrates the importance of testing for FLT3 mutations in all patients diagnosed with AML.
The context in which an FLT3 mutation occurs is quite important. I think its biggest prognostic impact is in the normal karyotype subtype, or what we used to call de novo AML. A frequent combination is mutations in both NPM1 and FLT3.9,20,21 If you see an NPM1 mutation, you should be looking for an FLT3 mutation. At a molecular level, this happens because NPM1 mutations lead to upregulation of FLT3 expression, so the cell wants to be dependent upon FLT3.
Guideline Recommendations for AML Molecular Testing
The American Society of Clinical Oncology/College of American Pathologists/American Society of Hematology guidelines recommend testing bone marrow or peripheral blood samples using fluorescence in situ hybridization, molecular genetics, and/or a next-generation sequencing (NGS) profile at diagnosis, the first sign of disease progression, and at relapse in patients with AML.22,23
Laboratory Testing for FLT3 Mutations
It is important to know how pathologists are handling the molecular tests for your patients. There is an FDA-approved companion assay for FLT3 inhibitors that can detect both FLT3-ITD and FLT3-TKD mutations, but that is not necessarily the assay your pathologist will use to analyze the sample. Many hospitals and academic centers may send samples to a local laboratory that performs its own PCR assay. Others send samples out to a commercial lab. PCR assays usually have a turnaround time of 3-5 days. PCR can detect FLT3-ITD mutations, but conventional PCR cannot detect FLT3-TKD mutations. FLT3-TKD can be detected by NGS, which can take 2-3 weeks to return or with the FDA-approved companion assay, which has a faster turnaround time. The difficulty with testing for FLT3 mutations is that the methods for detecting them differ.
PCR Testing for FLT3-ITD Mutations
For PCR testing of FLT3-ITD mutations, either bone marrow or peripheral blood may be used and it is essential to know how many blasts are being sent for analysis. If you send off an FLT3 test on patients with only 1% blasts in their marrow and who are in early relapse, there may be an FLT3 mutation, but you are unlikely to detect it because the test is not very sensitive.24,25
If you send a sample that is pure blasts, you have a very good idea of how much FLT3-ITD mutation is there. If a mutation is heterozygous, then only one half of the alleles in that sample have the mutation. Even when you are testing pure leukemia, there are still potential issues with PCR. The ITD insertions are variable in length, and the longer they are, the more difficult they are to detect by PCR. The allelic ratio is basically how much mutant gene there is relative to wild-type gene. If your ability to accurately quantify the amount of FLT3-ITD mutations in the sample is in question, then so is that ratio. There is no question that having a higher percentage of FLT3-ITD mutations is associated with a poor prognosis, but trying to identify a specific cutoff for a good vs bad allele ratio is not possible.
Furthermore, having any FLT3 mutation offers the potential for benefit from an FLT3 inhibitor. Although having lower levels is associated with a better prognosis, you should still strongly consider an FLT3 inhibitor. Therefore, you should never dismiss the presence of any FLT3 mutation. Just to reiterate: Although we are always interested in the frequency of mutation, our management does not usually change based on this frequency. A patient in early relapse with a 10% blast count might have an FLT3-ITD mutation result of only 2%. Although this might seem like nothing to worry about, it should not be ignored. It is probable that every leukemia cell has the mutation. Understanding what sample was sent for analysis and the number of blasts it has is critical for interpretation of the results.
Harry P. Erba, MD, PhD
I completely agree. Healthcare professionals may not always understand what happens in the laboratory with the genetic testing and some may think that pathologists use flow cytometry to isolate the blast population and then study only those cells. That is not the case. The entire sample is used for DNA extraction. As Dr Levis noted, the percentage of blasts in the sample is important. FLT3 mutations drive blast proliferation. FLT3 is never a mutation seen in clonal hematopoiesis of indeterminate prognosis. It is not an early event of clonal hematopoiesis.
Next-Generation Sequencing for FLT3 Mutation Testing
Mark J. Levis, MD, PhD
Originally NGS was not very accurate in detecting FLT3-ITD mutations because they are not mutant sequences, but normal sequences duplicated. The algorithms can be adjusted to improve accuracy, but the main concern is that it can take 2-3 weeks to get the results.26 FLT3 inhibitors need to be introduced relatively early in the course of disease. In addition, NGS will detect lots of noncanonical point mutations that have an unknown significance, so these data are not clinically useful.
One should not be using NGS as a standard way to detect FLT3-ITD mutations in patients with newly diagnosed AML. Instead, healthcare professionals should use PCR. Although FLT3-TKD mutations are less common, you may also want to have the ability to detect FLT3-TKD mutations more rapidly than using NGS, although that would take a specialized test or the use of the approved companion diagnostic.
Harry P. Erba, MD, PhD
Of note, not all NGS platforms accurately detect FLT3-ITD mutations with a length greater than 150 base pairs.27 This has led to some problems because local laboratories indicate that an FLT3-ITD mutation is present, but the NGS is not reporting it. This is why the PCR test is so important, and we act upon the knowledge that a patient has an FLT3-ITD mutation as detected by PCR.
Recommended Timelines for Initial AML Genetic Workup
Harry P. Erba, MD, PhD
Patients with FLT3-ITD–mutated disease typically present with white blood cell counts of more than 50,000, and the predominant cells are blasts. In these patients, you can send peripheral blood alone for the FLT3 PCR test. You do not have to wait days for a bone marrow biopsy.
Mark J. Levis, MD, PhD
That is an incredibly important point. If someone presents with a white blood cell count of 140,000, for example, and it is all blasts, you can be quite sure that this patient has AML. Flow cytometry can be used to make the diagnosis and in many cases, you do not need a bone marrow biopsy to diagnose this disease. You can send off blood samples for molecular testing when the patient initially presents, not 3-4 days later.
This is not something that you can just wait on for 3-4 weeks and then decide what to do.19 Optimal induction therapy for these patients depends on getting laboratory results back in the first week. You really do not have the luxury of waiting for a comprehensive NGS result, as it will likely come back outside the window where the patient may benefit from an FLT3 inhibitor.
Harry P. Erba, MD, PhD
In the current therapeutic landscape of AML, aside from PML::RARA fusion in APL, the FLT3-ITD is the most important mutation to identify early in the treatment course so that you can add an FLT3 inhibitor, which has been shown to improve patient survival when added to intensive chemotherapy.5,6
There are other FLT3 mutations that may affect treatment options, but there is more debate about the value of initial detection for these other FLT3 mutations. The FLT3-ITD mutation is the exception. If you are going to do 1 PCR test, this is the one you should do in a patient with newly diagnosed AML. As a reminder, you have to send in a sample that contains the blast, because this is the mutation driving the blast proliferation in the disease.
Key Takeaways on Molecular Testing
Mark J. Levis, MD, PhD
These are what I see as the important take-home points about testing:
Harry P. Erba, MD, PhD
Chemotherapy can start immediately after you take the sample for testing. Despite the patient undergoing chemotherapy, testing can be performed. If results are back indicating an FLT3 mutation by Day 8, then an FLT3 inhibitor can be added. That is when we add on the FLT3 inhibitor, whether it is quizartinib or midostaurin. The patient is receiving therapy and it gives you a week for PCR testing to be completed. But if you are only testing by NGS, you will not get that result back for at least a week, and you miss the time point when you can add the FLT3 inhibitor.
If the start of the FLT3 inhibitor is delayed, then you may not see the survival benefit that was shown in the clinical trials.5,6 In this case I recommend not continuing it for the entire 2 weeks but stopping it by Day 21 because FLT3 inhibitors can cause delays in blood count recovery.
Repeat FLT3 Mutation Testing Is Needed at Relapse
Harry P. Erba, MD, PhD
FLT3 mutations, especially FLT3-ITD mutations, are not the earliest events in leukemogenesis. FLT3-ITD mutations may be present in a subclone of the disease in a very small amount early on.28,29 If this is not detected and a patient receives chemotherapy and develops refractory disease, that subclone may grow and take over.
Patients receiving initial therapy for AML who are not achieving the desired response in their bone marrow still have highly populated blasts. Of importance, one cannot assume that it is the exact same disease at progression or relapse. The treatment may have selected for a new clone. There are multiple clones at the time of diagnosis that may still be present because they were resistant to chemotherapy.
In a patient who has received initial therapy for FLT3 wild-type AML but has persistent disease, do not forget to retest for an FLT3-ITD mutation because a clone may have emerged. The same is true for patients who relapse after responding to initial therapy, even if they did not have an FLT3-ITD mutation or FLT3-TKD mutation at diagnosis. These mutations may be present and have significant therapeutic implications, even in the setting of relapse.
Mark J. Levis, MD, PhD
A key point about this disease that we have learned and why you can never ignore even a small amount of an FLT3 mutant clone that is present at diagnosis is because it can drive a relapse or refractory state. Patients who have a small FLT3-ITD clone at diagnosis who are given standard chemotherapy without an FLT3 inhibitor may have that clone become undetectable, but it is probably still there. You should always recheck FLT3 mutation status when there is a change in clinical status, a relapse, or a refractory state. FLT3-ITD mutations occur in virtually all types of AML and can be present at diagnosis and absent at relapse or absent at diagnosis and present at relapse. Most of the time when it is present at diagnosis it will be present at relapse. The key is always to recheck at any change in clinical status and never ignore an FLT3 mutant clone if detected, no matter how small it is.
Additional Educational Activities on FLT3-Mutated AML
Watch for additional educational activities from Clinical Care Options to learn more about treating patients with FLT3 mutations and the management of FLT3 inhibitor treatment-related adverse events.
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