REVIEW ARTICLE

The clinical impact of the molecular landscape of acute

myeloid leukemia
Sabine Kayser1 and Mark J. Levis2
Correspondence: S. Kayser
s.kayser@dkfz-heidelberg.de
1
NCT Trial Center, National Center of Tumor Diseases, German Cancer Research Center
Received: May 28, 2022.
(DKFZ), Heidelberg, Germany and 2Sidney Kimmel Comprehensive Cancer Center, Johns
Accepted: July 28, 2022.
Hopkins University Baltimore, MD, USA
https://doi.org/10.3324/haematol.2022.280801

©2023 Ferrata Storti Foundation

Published under a CC BY-NC license

Abstract
Research into the underlying pathogenic mechanisms of acute myeloid leukemia (AML) has led to remarkable advances
in our understanding of the disease. Mutations now allow us to explore the enormous diversity among cytogenetically de-
fined subsets of AML, particularly the large subset of cytogenetically normal AML. Despite the progress in unraveling the
tumor genome, only a small number of recurrent mutations have been incorporated into risk-stratification schemes and
have been proven to be clinically relevant, targetable lesions. The current World Health Organization Classification of mye-
loid neoplasms and leukemia includes eight AML categories defined by recurrent genetic abnormalities as well as three
categories defined by gene mutations. We here discuss the utility of molecular markers in AML in prognostication and
treatment decision-making. New therapies based on targetable markers include IDH inhibitors (ivosidenib, enasidenib),
venetoclax-based therapy, FLT3 inhibitors (midostaurin, gilteritinib, and quizartinib), gemtuzumab ozogamicin, magrolimab
and menin inhibitors.

number have been incorporated into risk-stratification

Introduction schemes such as the National Comprehensive Cancer
Acute myeloid leukemia (AML) is a genetically heterogene- Network or European LeukemiaNet (ELN) guidelines.7,8
ous disease with identifiable somatic mutations in 97.3% Until recently most patients have been treated with simi-
of all cases.1 Besides age and comorbidities, the prognosis lar chemotherapeutic regimens.9 However, treatment op-
for patients with AML is largely determined by the biology tions for AML have expanded as a result of the discovery
of the disease.2 Targeted sequencing has identified several of genetic abnormalities. Since 2017, eight new targeted
mutations that carry prognostic information, including drugs have been approved by the Food & Drug Adminis-
mutations in FLT3, NPM1, KIT, CEBPA and TP53.3 In addi- tration (FDA) and six by the European Medicines Agency
tion, massively parallel sequencing led to the discovery of (EMA).10 These new therapeutics subsume tyrosine kinase
recurrent mutations in DNMT3A and IDH.4,5 Consistently, inhibitors (TKI), immune checkpoint inhibitors, mono-
the recurrent genetic abnormalities defining subtypes of clonal or bispecific T-cell engager antibodies, as well as
AML are associated with distinctive clinicopathological metabolic and pro-apoptotic agents. Targeting FLT3-ki-
features, impact prognosis, and are influencing treatment nase signaling is particularly important given that approxi-
choices. Thus, AML with recurrent genetic abnormalities mately one third of AML patients have a FLT3 mutation.3
is included within the current World Health Organization In this article, we give an overview of the clinical impact
(WHO) classification as a separate entity.6 AML with FLT3 of molecular markers in AML and how they are used as
is not included as a separate entity, because it occurs improved strategies for cancer therapy.
across multiple subtypes. However, the WHO classifica-
tion acknowledges that FLT3 mutations should be looked
for in all AML cases.6
Although new molecular analysis techniques such as
Isocitrate dehydrogenase
ultra-deep sequencing have helped to identify numerous Isocitrate dehydrogenase (IDH) is an enzyme that cata-
recurrent genetic abnormalities, to date, only a limited lyzes the oxidative decarboxylation of isocitrate to a-ke-

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REVIEW ARTICLE - Clinical impact of molecular markers in AML S. Kayser and M.J. Levis

toglutarate. Its two isoforms IDH1/IDH2 are recurrently the FDA for this group of patients in 2019.25
mutated in roughly 20% of de novo AML.3 Mutations in In addition, enasidenib was approved by the FDA in 2017
IDH1 occur in about 8% of AML patients and are almost for the treatment of relapsed/refractory AML with an IDH2
exclusively located at R132.1 IDH2 mutations can be de- mutation on the basis of a phase I/II trial.26 The overall re-
tected in almost 12% and involve substitutions at R140 or sponse rate (ORR) with enasidenib was 40.3% and the
R172.11 IDH mutations are frequently associated with in- median OS was 9.3 months. Enasidenib also showed
termediate-risk or normal karyotype cytogenetics.12,13 Gen- moderate efficacy in a phase I/II trial of 39 older patients
etically, IDH mutations are associated with NPM1 with newly diagnosed AML, resulting in a CR/CRi rate of
mutations,12-15 but less frequently co-occur with TET2 or 21%, an ORR of 30.8%, and a median OS of 11.3 months.27
WT1 mutations, which might be because all three classes However, primary and acquired resistance to these drugs
of mutations affect DNA methylation.16 Biologically, IDH are major clinical issues.28 Leukemia stemness seems to
mutations lead to increased levels of the oncometabolite be a major driver of primary resistance to IDH inhibitors,
2-hydroxyglutarate and consecutively result in arrest of whereas the selection of mutations in RUNX1/CEBPA or
hematopoietic differentiation via inhibition of histone de- RAS-RTK pathway genes seems to be the main driver of
methylation.17,18 acquired resistance, along with BCOR, homologous IDH
The data regarding outcomes of patients with IDH-mu- and TET2 mutations, as could be shown by sequencing
tated AML are conflicting. Three reports from cooperative analysis in serial samples from 60 IDH-mutated AML pa-
study groups showed a negative impact of cooperating tients treated with an IDH inhibitor.28
IDH1/2 mutations on relapse-free survival/relapse risk and While TET2 is directly affected by the 2-hydroxygluta-
overall survival (OS) in AML patients exhibiting the geno- rate-mediated oncometabolism, it is not yet clear how
type mutated NPM1 with unmutated FLT3-internal tandem loss-of-function mutations in BCOR, a transcription
duplication (ITD).14,15,19 In a retrospective analysis of 319 pa- corepressor,29 contribute to acquired resistance to IDH
tients with newly diagnosed, IDH-mutated AML (127 with inhibition.
IDH1, 135 with IDH2R140, and 57 with IDH2R172 mutations) In Europe, IDH inhibitors are currently not approved for
treated with intensive chemotherapy in three Acute IDH-mutated AML since the pharmaceutical company
Leukemia French Association (ALFA) prospective trials the could not fully address the major objections raised by the
presence of NPM1 mutations was the only variable pre- Committee for Medicinal Products for Human Use to sup-
dicting improved OS in multivariate analysis (P<0.0001).20 port a positive benefit/risk assessment in the proposed
In contrast, Patel et al. reported a favorable impact of the indication. Very recently, data from the global, randomized
genotype mutated NPM1 with unmutated FLT3-ITD only if double-blind phase III trial (AGILE) evaluating ivosidenib +
cooperating IDH1/2 mutations were present.21 The prog- azacitidine in patients with newly diagnosed AML with an
nostic significance of IDH2 mutations in AML also seems IDH1 mutation were published.30 Ivosidenib and azacitidine
to depend on the location of the mutation (IDH1: single significantly improved the CR rate (47.2% vs. 14.9%;
nucleotide polymorphism vs. R132;22 IDH2: R140 vs. 172).23 P<0.0001), event-free survival (hazard ratio [HR]=0.33,
The effects on survival are likely distinct for each of the P=0.002) and OS (24 months vs. 7.9 months, P=0.001) as
IDH mutations, with the presence or absence of other mu- compared to placebo + azacitidine in patients with newly
tations also affecting outcomes. Such opposing effects of diagnosed IDH1-mutated AML ineligible for intensive in-
genotypes on outcome highlight the statistical shortcom- duction chemotherapy. Based on these data, in March
ings of retrospective molecular studies. 2022 the pharmaceutical company submitted a marketing
Nevertheless, treatment with the IDH inhibitors enaside- authorization application to the EMA for ivosidenib in com-
nib and ivosidenib has added to the armamentarium of bination with azacitidine as first-line treatment in patients
targeted therapy. Currently, IDH inhibitors are approved by with previously untreated IDH1-mutated AML who are not
the FDA as treatment for relapsed/refractory AML with an eligible for intensive chemotherapy. In addition, on May 25,
IDH mutation as well as in newly diagnosed IDH1-mutated 2022 the FDA approved ivosidenib in combination with
AML patients not eligible for intensive chemotherapy. In azacitidine for newly diagnosed AML with an IDH1 muta-
2018, the approval of ivosidenib by the FDA was based on tion, as detected by an FDA-approved test in adults 75
an open-label, single-arm phase I trial showing a com- years or older, or who have comorbidities that preclude
plete response (CR)/complete response with incomplete the use of intensive induction chemotherapy.
count recovery (CRi) rate of 30.4% and a median OS of 8.8 Currently, a study of ivosidenib or enasidenib in combina-
months in patients with relapsed/refractory IDH1-mutated tion with induction and consolidation chemotherapy, fol-
AML.24 In the group of patients with newly diagnosed IDH1- lowed by maintenance therapy in patients with newly
mutated AML not eligible for intensive chemotherapy, a diagnosed AML or myelodysplastic syndrome (MDS) with
CR/CRi rate of 42.4% was achieved with a median OS of excess blasts 2 with an IDH mutation is also recruiting
12.6 months. Ivosidenib was subsequently approved by (HOVON150AML; NCT03839771).

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REVIEW ARTICLE - Clinical impact of molecular markers in AML S. Kayser and M.J. Levis

A high CR rate was also achieved in the VIALE-A study in (e.g. NCT04038437; V-FAST, NCT04075747) or standard in-
AML patients not eligible for intensive chemotherapy after tensive chemotherapy (e.g. NCT03709758; NCT04628026)
treatment with hypomethylating agents (HMA) and vene- is now being studied as frontline therapy in younger and
toclax,31 particularly in patients with IDH (CR+CRi rate: older patients with AML. Preliminary data suggest a very
75.4% vs. 10.7%; P<0.001) or NPM1 mutations within a nor- high ORR of 100% (n=10), with 75% (n=6/8) of the patients
mal karyotype (CR+CRi rate: 66.7 vs. 23.5%; P=0.012; achieving measurable residual disease (MRD)-negative re-
VIALE-A).32 Regarding durable remissions with responses missions assessed using multiparameter flow cytometry.
lasting for >12 months, NPM1 (9/18; 50%) and IDH2 (7/18; No dose-limiting toxicities were reported in the 200 or 400
39%) were among the most frequently mutated genes, mg dosing cohort, whereas one dose-limiting toxicity oc-
with survival ongoing after 21 to 49 months follow-up.32 curred in the 600 mg dose-escalation cohort (death due
Regarding IDH1-mutated patients, there was no difference to septic shock). Thus, 400 mg (the current FDA/EMA-ap-
in median OS between IDH1-mutated and wild-type pa- proved venetoclax dose for AML in combination with HMA)
tients (18.3 vs. 12.7 months; P=0.79). was determined to be the maximal tolerated dose in com-
Comparable results were achieved with low-dose cytara- bination with ‘7 + 3’ induction. The median time to count
bine (LDAC) and venetoclax in the VIALE-C study, although recovery (defined as an absolute neutrophil count
the trial failed to meet its primary endpoint of improved ≥0.5x109/L and a platelet count ≥50x109/L) following ‘7 +
OS with the addition of venetoclax to LDAC (7.2 months 3’ + venetoclax induction was 36 days.35 These results, if
vs. 4.1 months; HR=0.75; 95% confidence interval [95% CI]: confirmed in a larger number of patients, may soon indi-
0.52-1.07; P=0.11).33 However, in an unplanned analysis with cate the new standard of care for younger, intensively
an additional 6 months of follow-up a significantly su- treatable patients.
perior median OS of 8.4 months for venetoclax in com- Venetoclax in combination with other agents is also being
bination with LDAC (HR=0.70; 95% CI: 0.50-0.98; P=0.04) evaluated in relapsed/refractory AML patients (e.g. gem-
as compared to 4.1 months after LDAC + placebo as well tuzumab ozogamicin [GO] + venetoclax, NCT04070768; gil-
as overall response (48% vs. 13%; P<0.001) and CR rates teritinib + venetoclax, NCT03625505; NCT04330820; oral
(27% vs. 7%; P<0.001) were achieved.33 azacitidine + venetoclax, NCT04887857). Additionally,
Based on these results, the FDA and EMA have approved venetoclax in combination with azacitidine is being evalu-
venetoclax for newly diagnosed AML patients ≥75 years ated in MRD-positive AML/MDS patients after allogeneic
old or ineligible for intensive chemotherapy in combina- stem cell transplantation (allo-SCT) (NCT04809181) as well
tion with HMA or LDAC. Currently, these combinations of as in patients with molecular relapse/progression of
HMA or LDAC with venetoclax are standard of care in NPM1-mutated AML (NCT04867928).
older/unfit patients with AML. There are no clear data to Based on the above data, patients with IDH1 mutations
support the superiority of one HMA over another, although should be treated with ivosidenib (+ azacitidine), whereas
there are more data with the azacitidine combinations. for patients with IDH2, venetoclax in combination with
Recently, results from a phase Ib trial evaluating veneto- azacitidine currently seems the best option, at least in
clax in combination with standard chemotherapy in 51 older patients. For younger patients, who are eligible for
elderly AML patients (median age, 72 years; range, 63-80 intensive chemotherapy, treatment with intensive chemo-
years) were published.34 During induction, a 7-day pre- therapy in combination with ivosidenib (IDH1-mutated) or
phase/dose ramp-up (days -6 to 0) was followed by an venetoclax (IDH2-mutated) might be an option as soon as
additional 7 days of venetoclax combined with cytarabine more mature data are available.
100 mg/m2 intravenously on days 1-5 and idarubicin 12
mg/m2 intravenously on days 2-3 (i.e., “5 + 2”). Consolida-
tion chemotherapy (4 cycles) included 14 days of veneto-
clax (days -6 to 7) combined with cytarabine (days 1-2) and
FLT3 mutations
idarubicin (day 1). Maintenance venetoclax was permitted Approximately one third of AML patients harbor activating
(7 cycles). The combined chemotherapy with venetoclax FLT3 mutations, which lead to constitutive activation of a
was safe and tolerable, leading to an ORR (CR/CRi) of 72% receptor tyrosine kinase.3 Given the high incidence of
in fit older patients with AML. Patients with de novo AML these mutations, they are attractive targets for small-mol-
benefited particularly, with an ORR of 97% as compared ecule inhibition.36 Currently, only two FLT3 inhibitors are
to 43% in patients with secondary AML. The median OS approved by the FDA and EMA, midostaurin and gilteriti-
for the entire cohort was 11.2 months (95% CI: 7.3-20.1 nib. Midostaurin was the first approved TKI for use in com-
months). Patients with de novo AML had a longer median bination with standard intensive chemotherapy for adult
OS compared to those with secondary AML (31.3 vs. 6.1 patients without age restriction with newly diagnosed
months; P=0.0001).34 Given these promising results, vene- FLT3-mutated AML in the USA and Europe.37,38 The approval
toclax in combination with daunorubicin and cytarabine of midostaurin was based on the positive results of the

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large, international randomized phase III CALGB RATIFY agent maintenance therapy for 12 months in adult (median
trial.39 The addition of midostaurin to intensive chemo- age, 54 years; range, 18-70, 30% older than 60 years) AML
therapy significantly improved OS in younger adults with patients with FLT3-ITD showed that midostaurin in com-
FLT3-mutated AML with a median OS of 74.7 months for bination with intensive chemotherapy including allo-SCT
the midostaurin arm (range, 31.5 months - not reached) can be safely administered, also in older AML patients.44
as compared to 25.6 months for the placebo arm (range, In contrast to the RATIFY trial, in which midostaurin main-
18.6-42.9 months). Interestingly, this improvement was re- tenance therapy was only applied after high-dose cytara-
gardless of the FLT3 mutational status (either ITD or tyro- bine consolidation, midostaurin maintenance therapy was
sine kinase domain [TKD]) or the FLT3-ITD allelic ratio. also administered after allo-SCT. The landmark analysis at
Furthermore, patients undergoing allo-SCT in first CR had day 100 after transplantation favored maintenance therapy
a better outcome if they were treated with midostaurin after allo-SCT with better event-free survival and OS in
during induction therapy (P=0.08), suggesting that the op- patients starting maintenance therapy within 100 days
timal treatment strategy in FLT3-mutated AML would be after transplantation.44 Further evidence came from the
to move on to allo-SCT early in first CR.39 Given the re- RADIUS trial, evaluating midostaurin maintenance after
markable difference in survival after allo-SCT early in first allo-SCT.45 Inhibition of FLT3 phosphorylation to <70% of
CR in patients treated with midostaurin as compared to baseline (achieved by 50% of the midostaurin-treated pa-
those treated with placebo it seems that the combination tients) was associated with improved relapse-free sur-
of midostaurin with intensive chemotherapy results in vival. Thus, the addition of midostaurin maintenance
deeper remissions. The first evidence of this came from a therapy following allo-SCT may provide clinical benefit in
small translational study evaluating the level of MRD in 17 those FLT3-ITD patients with at least 70% inhibition of
patients treated with cytarabine and anthracycline-based phosphorylation.45 While this provides evidence that in-
intensive induction chemotherapy.40 A TKI was given to hibition of FLT3 as post-transplant maintenance therapy
eight (47%) of the 17 patients during induction therapy. In is of value, midostaurin does not seem to be the drug of
all cases, samples were evaluated at diagnosis and at re- choice, given that there is no way to predict who will
mission by a highly sensitive combination polymerase achieve adequate in vivo inhibition with this compound.
chain reaction next-generation sequencing MRD assay for More importantly, these data underline the need for ran-
FLT3-ITD. In those patients who were treated with chemo- domized trials to establish the concept of maintenance
therapy in combination with a TKI during induction, the with targeted agents after consolidation therapy to pre-
average level of ITD mutation was significantly lower than vent AML recurrence.
that in the nine patients treated with chemotherapy alone Biologically, maintenance with a TKI to inhibit FLT3 signal-
(P=0.008).40 ing seems to be reasonable, particularly in light of the
Of note, despite inclusion of maintenance therapy on the positive results from the SORMAIN,46 RADIUS45 and AD-
RATIFY protocol, the FDA did not approve midostaurin as MIRAL47 trials.
maintenance therapy, whereas the EMA included mainten- Gilteritinib, a novel, highly selective, potent oral FLT3 in-
ance in the drug’s product information.41 Lack of re-ran- hibitor with activity against ITD and TKD mutations, is the
domization prior to maintenance was cited by the FDA as only FDA- and EMA-approved TKI for the treatment of re-
a major reason; thus, the contribution of maintenance lapsed/refractory FLT3-mutated AML in the USA and Eu-
therapy to the treatment effect could not be determined.42 rope.48 The ideal dose of gilteritinib was identified from
Results from a post-hoc subset analysis of the RATIFY trial the multicenter, open-label phase I/II Chrysalis trial evalu-
demonstrated no difference in the disease-free survival ating 252 FLT3-mutated patients. Gilteritinib resulted in
between the treatment arms during the 12 cycles of main- prolonged responses in patients with heavily pre-treated,
tenance (P=0.49) and no difference in OS from the time of relapsed or refractory AML.49 The ORR was 40%, with 8%
starting maintenance (P=0.35).43 Nevertheless, the data achieving CR, 4% CRi, 18% CR with incomplete hemato-
suggest that midostaurin may have delayed, but not pre- logic recovery (CRh), and 10% partial remission. The
vented relapse in some of these patients, since more re- median OS was 25 weeks (95% CI: 20-30 weeks) and
lapses were observed after stopping midostaurin (17/69 median duration of response 17 weeks (95% CI: 14-29
[25%] vs. 7/51 [14%] on the placebo arm), and more of weeks).49 In addition, gilteritinib was evaluated within a
these relapses occurred within the first 6 months (14 randomized, open-label, multicenter phase III trial (AD-
[20%] vs. 2 [4%]).43 Unfortunately, patients were not re- MIRAL trial) of relapsed/refractory FLT3-mutated patients,
randomized at the start of the maintenance treatment. who were randomized 2:1 to receive gilteritinib or salvage
Moreover, the numbers were too small for any clinically chemotherapy.50 Salvage chemotherapy options were
meaningful comparisons. Recently published results of a LDAC, azacitidine, mitoxantrone/etoposide/cytarabine, or
phase II trial evaluating midostaurin in combination with fludarabine/cytarabine/idarubicin and granulocyte colony-
intensive chemotherapy followed by allo-SCT and single- stimulating factor (FLAG-IDA). Randomization was strat-

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ified by response to first-line AML therapy and pre-spec- was noted, venetoclax was stopped. Encouraging results
ified chemotherapy (intensive vs. low-intensity). The CR + were achieved with an ORR (CR+ CRi + morphologic leuke-
CRh rate was 21%; the median time to response was 3.6 mia-free state) of 67% in relapsed/refractory patients and
months (range, 0.9-9.6 months) and the median duration 100% in newly diagnosed patients (CR/CRi, n=9, 82%).53
of response was 4.6 months.50 The median OS was signifi- Currently, several trials of gilteritinib are underway, includ-
cantly longer after gilteritinib than in the salvage chemo- ing a trial of gilteritinib versus placebo as maintenance
therapy arm (9.3 months vs. 5.6 months), and 37.1% therapy after consolidation (NCT02927262) or after allo-
compared to 16.7% of the patients were alive at 12 SCT in patients with FLT3-ITD mutations (NCT02997202).
months.50 Furthermore, the OS benefit was observed in There are ongoing trials combining gilteritinib with atezol-
patients preselected for both high-intensity (HR=0.66, izumab (NCT03730012) and venetoclax (NCT03625505) in
95% CI: 0.47-0.93) and low-intensity chemotherapy patients with relapsed/refractory AML as well as a ran-
(HR=0.56, 95% CI: 0.38-0.84).50 Overall, the results support domized phase II trial of gilteritinib versus midostaurin in
the use of gilteritinib in patients with relapsed/refractory combination with induction and consolidation chemother-
AML. Based on these results, gilteritinib is now approved apy (NCT03836209).
by the FDA and EMA for the treatment of relapsed/refrac- High CR rates have also been observed with standard in-
tory patients with FLT3-mutated AML. duction therapy with the “7+3” regimen combined with
Recently, results from the phase III randomized trial com- crenolanib (72%)54 and quizartinib (84%),55 surpassing the
paring gilteritinib in combination with azacitidine versus 59% CR rate observed within the RATIFY trial.39 In addition,
azacitidine monotherapy for adult patients with newly di- quizartinib (40 mg/day on days 8-21) versus placebo in
agnosed FLT3-mutated AML ineligible for standard chemo- combination with standard induction and post-remission
therapy were presented (LACEWING trial, NCT02752035).51 therapy (including allo-SCT) followed by up to 3 years of
While gilteritinib + azacitidine led to significantly higher maintenance therapy with quizartinib (30-60 mg/day) or
composite CR rates, the combined therapy failed to im- placebo was evaluated in 539 adult patients with FLT3-
prove OS compared to azacitidine alone. Thus, an inde- ITD AML within the global, randomized, double-blind,
pendent data monitoring committee recommended placebo-controlled phase III QuANTUM-First trial
terminating the study for futility, since concluding results (NCT02668653).56 Therapy with quizartinib resulted in a
are unlikely to show a statistically significant increase in significantly longer median OS of 31.9 months as com-
OS. As a consequence, the pharmaceutical company has pared to 15.1 months with placebo. The safety of quizarti-
stopped enrollment. nib was shown to be manageable and consistent with the
In addition, results from a phase I/II study evaluating gil- known safety profile. Thus, the pharmaceutical company
teritinib combined with the “7+3” regimen and consolida- will share the data with regulatory authorities for possible
tion treatment were presented at the annual meeting of global approval. Currently, quizartinib is approved in Japan
the European Hematology Association.52 In this dose-es- as treatment for adult patients with relapsed/refractory
calation study, 68 FLT3-mutated AML patients received FLT3-ITD mutated AML (approval granted in October 2019).
gilteritinib (40, 80, 120, or 200 mg/day) in one of two While these results are premature, the high CR rates sug-
schedules, in combination with the “7+3” regimen (sched- gest that the benefit of selective FLT3 inhibitors is not just
ule 1: days 4-17; schedule 2: days 8-21). The maximum tol- in depth of response, as with midostaurin, but that more
erated dose of gilteritinib was determined to be 120 mg, patients may respond overall.
which was evaluated in a dose expansion cohort. Remark- Nevertheless, although AML patients may respond to FLT3
ably, the composite CR rate of FLT3-mutated patients re- inhibitors, the duration of the response is still mostly short
ceiving gilteritinib on schedule 1 (n=22) was 100% and on due to primary and acquired resistance. The most com-
schedule 2 (n=11) 81.8% with a median disease-free sur- mon mechanism of resistance is due to acquired FLT3-TKD
vival of 297 days.52 Very promising results were also mutations, such as F691L and D835.57 These mutations
achieved in a phase I/II trial in 26 patients with either re- hinder TKI binding and lead to an active kinase conforma-
lapsed/refractory FLT3-mutated AML or high-risk tion.58,59 This mechanism of resistance was reported for
MDS/chronic myelomonocytic leukemia (n=15) or patients type II inhibitors, including quizartinib. In contrast, gilte-
with newly diagnosed FLT3-mutated AML (n=11) unsuitable ritinib and crenolanib showed clinical activity against
for intensive chemotherapy.53 All patients received azaciti- FLT3-TKD D835 mutations. Nevertheless, the data suggest
dine 75 mg/m2 subcutaneously/intravenously on days 1-7, that this is mainly the case if the FLT3-TKD mutation co-
venetoclax for up to 28 days, and gilteritinib on days 1-28. exists with FLT3-ITD, but not as much if FLT3-TKD D835 is
The gilteritinib dose ranged from 80 mg to 120 mg daily the only mutation.60 Moreover, they had limited activity
during the phase I dose escalation (3+3 design). However, against F691L mutations.61 Further mechanisms of resis-
the triple therapy was so myelosuppressive that a bone tance are related to the acquisition of multiple RAS/MAPK
marrow evaluation was performed on day 14 and if aplasia pathway gene mutations at relapse, frequently in alter-

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native clones, suggesting a high level of pathway reacti- nostic impact of NPM1 mutations should be interpreted in
vation.62 the context of a FLT3-ITD, which occurs in roughly 45% of
Recently, the selective and irreversible FLT3 inhibitor, FF- normal karyotype AML.1,71-74 Particularly in NPM1-mutated
10101, was found to have significant activity against FLT3- patients with a concurrent high FLT3-ITD allelic ratio
ITD and -TKD mutations, including F691L and D835, both (≥0.5)8,75-77 the favorable prognostic effect of NPM1 is miti-
in vitro and in vivo.63,64 Thus, the inhibitor was evaluated in gated or even abolished as compared to that in patients
a phase I dose escalation study in 52 patients with refrac- with a low allelic ratio.76,77 In comparison, patients with
tory/relapsed AML.65 Continuous treatment with FF-10101 mutated NPM1 without FLT3-ITD or FLT3-ITD with a low
at a dose of 10-225 mg four times a day or 50-100 mg allelic ratio (<0.5) have a somewhat better outcome.8,77
twice daily in pretreated patients (median number of prior These data have recently been confirmed in a large cohort
therapies, n=3) resulted in a composite CR rate of 13% and of intensively treated adult AML patients.78 Moreover,
a partial response rate of 8%, including those with acti- IDH1/2 mutations may also exert a negative prognostic im-
vating FLT3-TKD mutations resistant to gilteritinib and pact on relapse-free survival and OS in patients with mu-
other FLT3 kinase inhibitors.65 Doses of 50-75 mg twice tated NPM1 without FLT3-ITD.14,15 In a retrospective analysis
daily were well tolerated and resulted in sustained FLT3 of 319 patients with newly diagnosed AML and an IDH mu-
inhibition. The trial is currently active, but not recruiting tation (127 with IDH1, 135 with IDH2R140, and 57 with IDH2R172
patients (NCT03194685). mutations) treated with intensive chemotherapy in three
We suggest treatment with gilteritinib for relapsed/refrac- Acute Leukemia French Association prospective trials the
tory FLT3-mutated AML patients, particularly in those pa- presence of NPM1 mutations was the only variable pre-
tients not eligible for intensive therapy strategies. In dicting improved OS in multivariate analysis (P<0.0001).20
younger relapsed/refractory patients, gilteritinib could be In contrast, Patel et al. reported on a favorable impact of
used as bridge to transplant. Based on the QuANTUM-First mutated NPM1 without FLT3-ITD only if cooperating IDH1/2
data, quizartinib in combination with standard chemo- mutations were present.21 Such opposing effects of geno-
therapy might soon be available as intensive first-line types on outcome highlight the statistical shortcomings
treatment in younger patients. of retrospective molecular studies.
Whether patients with NPM1-mutated or core binding fac- In a study of 245 adult patients with NPM1-mutated AML,
tor-rearranged (both CD33-positive), newly diagnosed AML relevant MRD checkpoints could be defined.79 Achiev-
and a FLT3 mutation might benefit from combined therapy ement of RT-qPCR negativity after two courses of induc-
(midostaurin + GO + standard “7+3” chemotherapy) is cur- tion therapy identified patients with a low cumulative
rently being evaluated in a phase I/II trial (MOSAIC trial, incidence of relapse (6.5% after 4 years) as compared to
NCT04385290). that of RT-qPCR-positive patients (53% after 4 years;
P<0.001), translating into significant differences in OS (90%
vs. 51%, respectively; P=0.001). After completion of therapy,
Acute myeloid leukemia with the cumulative incidence of relapse was 15.7% in MRD-
negative patients as compared to 66.5% in MRD-positive
mutated NPM1 patients (P<0.001).79 Another study indicated that a NPM1
NPM1 mutations are one of the most frequent molecular mutation cut-off level of 0.01 after induction therapy, as
abnormalities in AML, particularly in patients with a nor- measured by RT-qPCR (with a sensitivity of 10−6), was as-
mal karyotype.1 NPM1 mutations result in cytoplasmic ac- sociated with a cumulative incidence of relapse after 2
cumulation of the protein, although it is presently still years of 77.8% for patients with ratios above the cut-off
unclear how they contribute to leukemic transformation.66 as compared to 26.4% for those with ratios below the cut-
The NPM1 mutations subtypes A, B, and D comprise 90% off. In addition, NPM1 MRD positivity by RT-qPCR before
of all variants. These three mutation subtypes have been allo-SCT is independently associated with a significantly
shown to be reliable markers for MRD detection with high increased risk of relapse and inferior survival.81,82 Assuming
sensitivity.67,68 To date, however, more than 50 different that a further reduction of MRD levels optimizes outcome
NPM1 mutations have been reported.69 The same assay can after allo-SCT, this relationship would justify risk-stratified
be adapted for cases with rare NPM1 mutation variants by treatment allocation, including the use of additional pre-
replacing mutation-specific primers, but case-specific transplant chemotherapy. However, as MRD might simply
quantitative, reverse transcriptase polymerase chain re- reflect reduced sensitivity of leukemia cells to chemother-
actions (RT-qPCR) need to be carefully established to apy, the presence of MRD might only mark those patients
avoid non-specific background amplification from the who are unlikely to be cured with subsequent similar-type
wild-type NPM1 allele.70 therapies, even if disease levels are brought temporarily
In NPM1-mutated AML, concurrent mutations typically below the level of detection. Therefore, a further approach
occur in FLT3, DNMT3A, IDH1/2 or TET2.1 Thus, the prog- could be pre-emptive immune or antibody therapy

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REVIEW ARTICLE - Clinical impact of molecular markers in AML S. Kayser and M.J. Levis

(NCT02789254) in MRD-positive patients, such as pem- to venetoclax + HMA treatment with 2-year OS rates of
brolizumab and azacitidine (PEMAZA trial, NCT03769532) 71.8% and 79.5%, respectively.32 Similar results were docu-
or venetoclax and azacitidine (VIALE-M trial, mented within the phase III trial VIALE-A of venetoclax +
NCT04102020). HMA in newly diagnosed AML.31 Thus, elderly patients with
Increasing levels of NPM1 MRD were also predictive of an NPM1 mutations or patients not eligible for intensive treat-
impending relapse after chemotherapy (MRD increase >1% ment should be treated with venetoclax in combination
NPM1mut/ABL1) or allo-SCT (MRD increase >10% with azacitidine. NPM1-mutated AML also appears to be
NPM1mut/ABL1).83 Importantly, MRD status has been found responsive to menin inhibitors (see below).86
to be a better predictor of relapse risk than FLT3-ITD in
NPM1-mutated AML.84
In the randomized French ALFA-0701 trial showing the su-
periority of intensive chemotherapy in combination with
CD33-positive acute myeloid leukemia
GO over intensive chemotherapy alone NPM1-MRD was CD33 is highly expressed on cells of myeloid lineage, thus
predictive for response to therapy since more MRD- making it an attractive therapeutic target.87 GO is a hu-
negative results were obtained in patients treated in the manized anti-CD33 monoclonal antibody linked to the
GO arm than in those treated in the control arm after in- cytotoxic agent calicheamicin.88 GO initially received ac-
duction therapy (39% vs. 7%; P=0.006) as well as at the celerated approval from the FDA in 2000 for the treatment
end of treatment (91% vs. 61%; P=0.028).85 In addition, of patients with CD33-positive AML in first relapse who
positive NPM1-MRD (defined as >0.1% in the bone marrow) were ≥60 years and not suitable for intensive chemother-
after induction and at the end of treatment also predicted apy.89,90 However, GO was voluntarily withdrawn from the
a higher risk of relapse, but did not influence OS.85 market by the pharmaceutical company in 2010 when a
Patients with NPM1 or IDH2 mutations respond very well phase III trial comparing standard induction chemotherapy

Figure 1. Genes recurrently mutated in acute myeloid leukemia as well as mechanism of action of targeted therapies. TCA:
tricarboxylic acid cycle; IDH: isocitrate dehydrogenase; aKG: alpha-ketoglutarate; 2HG: 2-hydroxyglutarate.

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(“7+3”) with or without GO in patients younger than 60 of AML with TP53 alterations.101 Although responses were
years showed an increased early mortality rate (6% vs. not durable, they resulted in OS rates that were similar to
1%).91 Nevertheless, the early mortality rate in the standard those of AML patients with an intermediate-risk cytogen-
arm was unexpectedly low. Consecutively, three other ran- etic profile and who also received serial 10-day courses of
domized trials showed improved OS rates with the addi- decitabine.101
tion of GO in patients with favourable- and Moreover, in 55 patients with TP53-mutated MDS or AML,
intermediate-risk cytogenetics without increased induc- APR-246 (eprenetapopt), a novel, first-in-class, small mol-
tion mortality.92-94 A reduced risk of relapse (P=0.0001) and ecule, in combination with azacitidine led to an ORR of
improved survival (P=0.01) without increased rates of in- 73% with a CR rate of 50% in MDS patients (n=20/40) and
duction mortality in patients with favorable- and inter- of 64% and 36% in AML patients (n=4/11). The median sur-
mediate-risk cytogenetics were reported in a vival was 10.8 months with a significant improvement in
meta-analysis of five trials including 3,325 AML patients responding versus non-responding patients by landmark
randomized to receive GO along with intensive induction analysis (14.6 vs. 7.5 months; P=0.0005).102 Overall, 35%
chemotherapy.95 Thus, GO was reapproved by the FDA in (n=19/55) of the patients underwent allo-SCT with a
2017 and by the EMA in 2018 for the treatment of adult pa- median OS of 14.7 months. APR-246 was also evaluated in
tients (EMA: aged 15 years and older) with newly diag- a phase II trial in 52 TP53-mutated patients (34 with MDS,
nosed CD33-positive AML. In addition, GO is licenced in 18 with AML [including 7 with >30% blast cells]). The ORR
the USA as monotherapy for the treatment of patients among the patients with MDS was 62%, including a CR
aged 2 years and older with relapsed or refractory CD33- rate of 47%, with a median response duration of 10.4
positive AML as well as in patients with newly-diagnosed months. The ORR among those with AML was 33%, includ-
AML. ing a CR rate of 17% (27% and 0% CR in the AML patients
Thus, we suggest the use of GO in combination with in- with less than and more than 30% bone marrow blast
tensive chemotherapy in patients with CD33-positive, fa- cells, respectively). The main treatment-related adverse
vorable-risk AML (according to risk-stratification schemes events were febrile neutropenia (36%) and neurological
such as the National Comprehensive Cancer Network7 or events (40%), the latter correlating with a lower glomerular
ELN8 guidelines). filtration rate at treatment onset (P<0.01) and higher age
(P=0.05), and resolving with temporary drug interruption
without recurrence after adequate APR-246 dose reduc-
Acute myeloid leukemia with TP53 tion. With a median follow-up of 9.7 months, the median
OS was 12.1 months in MDS, and 13.9 and 3.0 months in
mutations AML with less than and more than 30% marrow blasts, re-
The tumor protein p53 (TP53) encodes a transcription fac- spectively.103 Recently, a randomized phase III trial of APR-
tor that is involved in cell cycle arrest and apoptosis.96 246 in combination with azacitidine versus azacitidine
TP53 mutations occur in roughly 12% of AML patients,97 alone in TP53-mutated MDS completed accrual
predominantly in therapy-related or secondary AML as (NCT03745716); the final results are pending. Additionally,
well as in elderly patients.98 Moreover, TP53 alterations are novel doublet and triplet therapy with venetoclax and aza-
found in roughly 70% of AML patients with a complex ka- citidine in combination with APR-246 (NCT04214860, com-
ryotype.99 TP53 mutations predict for very low CR rates pleted, final results are pending) or as post-transplant
(less than 30%) and were shown to be an independent maintenance are being investigated (NCT03931291, com-
poor prognostic factor among the subgroup of AML with pleted, final results are pending).
complex karyotype.99 Interestingly, TP53 could be ident- Recent data from two phase I trials suggest a high re-
ified in hematopoietic stem and progenitor cells in sponse rate after combination therapy with venetoclax
chemotherapy-naïve controls and in therapy-related or and decitabine, azacitidine104 or low-dose cytarabine105 in
secondary AML patients years prior to development of newly diagnosed elderly (≥60 years) AML patients not eli-
overt disease, suggesting that hematopoietic stem and gible for intensive chemotherapy, a group in whom a high
progenitor cells carrying TP53 may be chemotherapy-re- incidence of TP53 mutations would be suspected. Re-
sistant and expand after treatment.100 Individuals with sponses were also achieved in newly diagnosed patients
clonal hematopoiesis with indeterminate potential have a with TP53 mutations after treatment with venetoclax/aza-
13-fold increased risk of developing a hematologic malig- citidine within the VIALE-A trial, although these were
nancy, and this risk may be increased in the context of mostly short-lived and not durable.31
cytotoxic therapy, at least if a TP53 mutation is present.100 Although these data seem to be promising, durable re-
Recently published data suggest that treatment with deci- sponses are seldom observed. Thus, new treatment ap-
tabine at a dose of 20 mg/m² per day for 10 consecutive proaches are urgently needed for these very high-risk
days in monthly cycles may improve the dismal outcome patients.

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REVIEW ARTICLE - Clinical impact of molecular markers in AML S. Kayser and M.J. Levis

CD47-positive acute myeloid leukemia Lysine-specific methyltransferase 2A

CD47 is a “do not eat me” signal, strongly expressed in solid (KMT2A)
tumors and myeloid malignancies, which enables cells Mutations in the KMT2A gene (formerly known as Mixed
carrying this protein to evade macrophages.106,107 In preclini- Lineage Leukemia, MLL) at 11q23 occur in roughly 10% of
cal models of AML and MDS, it could be demonstrated that acute leukemias.114 KMT2A encodes a histone methyltrans-
blocking CD47 enhances antitumor response108,109 and that ferase, which regulates homeobox genes affecting hema-
anti-CD47 antibodies stimulate antibody-dependent cellu- topoiesis.115
lar phagocytosis, promoting priming and memory re- Menin is essential for the proliferation and survival of
sponses of CD8 T cells.110 KMT2A-rearranged and NPM1-mutated AML.116,117 NPM1 mu-
Magrolimab targets CD47 on tumor cells, including macro- tations have also been associated with the upregulation of
phage phagocytosis.10 Magrolimab is currently being evalu- HOXA genes, similar to gene expression patterns observed
ated in several early clinical trials in AML (e.g. in patients with KMT2A rearrangements.118,119 These findings
NCT04435691). Recently, a phase I trial of magrolimab with have led to the hypothesis that AML patients with NPM1
azacitidine documented an ORR of 91% in patients with mutations might also benefit from menin inhibition. Menin
MDS and a CR rate of 42%.111 In addition, high response rates inhibitors are a novel class of agents targeting the under-
were observed in TP53-mutated MDS patients lying biology of NPM1- and KMT2A-mutated AML. In pre-
(NCT03248479). Of note, AML patients with a TP53 mutation clinical models, small-molecule inhibitors of the
(n=12) showed a CR/CRi rate of 75%. With a median follow- menin-KMT2A protein-protein interaction induce differ-
up of 8.8 months, the median duration of response or OS entiation, downregulate critical gene expression programs,
was not met.112 Overall, the therapy was well tolerated, and and confer a survival advantage in patient-derived xeno-
no treatment-related febrile neutropenia occurred. Com- graft models of NPM1- and KMT2A-mutated AML.120-122 Four
mon treatment-related adverse events were anemia (44%), different menin-MLL1 inhibitors (SNDX-5613, NCT05326516;
fatigue (18%), infusion reactions (18%), neutropenia (8%) JNJ-75276617, NCT04811560; KO-539, NCT04067336; and
and thrombocytopenia (5%). In addition, no patient discon- DSP-5336, NCT04988555) are currently in early-phase
tinued treatment due to an adverse event. The mean de- clinical trials. Preliminary results in relapsed/refractory
cline in hemoglobin levels with the first dose of magrolimab AML with NPM1 and KMT2A mutations have shown prom-
+ azacitidine was 0.4 g/dL. Fifty-eight percent of the pa- ising clinical activity.123 In particular, the ORR after single-
tients no longer required red blood cell transfusions.112 En- agent therapy with SNDX-5613 in heavily pretreated
couraging results were also reported in a single-arm phase patients with relapsed/refractory NPM1- or KMT2A-mu-
Ib trial of magrolimab + azacitidine at the European Hema- tated AML was 55% with a composite CR MRD-negative
tology Association meeting 2022 in 72 TP53-mutated AML rate of 31% (n=16/51).86 In addition, responses were durable,
patients, unsuitable for intensive chemotherapy.113 Magroli- lasting more than 6 months in six of the 12 patients who
mab + azacitidine produced durable responses with an ORR achieved a composite CR.86 Finally, the highly selective, ir-
of 48.6% (CR, 33.3%; CRi/CRh, 8.3%; morphological leuke- reversible menin-inhibitor BMF-219 is currently being
mia-free state, 1.4%; partial response, 5.6%) and an encour- evaluated in a multicenter phase I trial in relapsed/refrac-
aging OS of 10.8 months (95% CI: 6.8-12.8 months). The tory patients with acute leukemia, diffuse large B-cell lym-
combined therapy had an acceptable safety profile, al- phoma or multiple myeloma (NCT05153330).
though there were some safety concerns due to anemia
(overall 29.2%; grade 3, 26.4%; grade 4, 2.8%). The most
common grade ≥3 adverse events were febrile neutropenia
(37.5%), thrombocytopenia (29.2%), pneumonia (26.4%), and
Conclusions
neutropenia (20.8%). Grade 3 hemolysis was reported in Progress in unraveling the molecular pathogenesis of AML
one patient (1.4%); no grade 4 hemolysis was reported. Ad- and the identification of the genetic determinants of re-
verse events led to discontinuation of magrolimab in 30.6% sponse to treatment have been impressive and translation
of the patients and of azacitidine in 29.2% of the patients. of these findings into clinical decision-making has been
Patients who proceeded to allo-SCT had an encouraging 1- increasing in recent years. The availability of a molecular
year OS of 63%.113 These observations are being further profile enables targeted-based treatment (Figure 1). The
evaluated in a randomized, open-label phase III trial for interplay between various molecular abnormalities sug-
adult AML patients with TP53 mutations (ENHANCE-2, gests that the combined inhibition of several signaling
NCT04778397) as well as a randomized phase III study of pathways is required to achieve maximum clinical benefit.
triple combination therapy with venetoclax/azacitidine + Thus, evaluation of the genetic profile at diagnosis, but
magrolimab versus venetoclax/azacitidine + placebo also at relapse is mandatory in all AML patients. Open
(NCT05079230). questions remain about whether patients should be

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REVIEW ARTICLE - Clinical impact of molecular markers in AML S. Kayser and M.J. Levis

treated with intensive or non-intensive approaches and Jazz Pharmaceuticals. MJL receives research funding from
how best to incorporate maintenance therapy, monoclonal Novartis and Astellas. MJL serves as a consultant for No-
antibodies as well as immunotherapy. vartis, Daiichi-Sankyo, Astellas, and Arog.

Disclosures Contributions
SK has served as a consultant for Novartis, Pfizer, Gilead and SK and MJL wrote the manuscript.

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