Gene expression profiling identifies FLT3 mutation-like cases in wild-type FLT3 acute myeloid leukemia

Adrián Mosquera Orgueira; Andrés Peleteiro Raíndo; Miguel Cid López; Beatriz Antelo Rodríguez; José Ángel Díaz Arias; Roi Ferreiro Ferro; Natalia Alonso Vence; Ángeles Bendaña López; Aitor Abuín Blanco; Laura Bao Pérez; Paula Melero Valentín; Marta Sonia González Pérez; Claudio Cerchione; Giovanni Martinelli; Pau Montesinos Fernández; Manuel Mateo Pérez Encinas; José Luis Bello López

doi:10.1371/journal.pone.0247093

. 2021 Feb 16;16(2):e0247093. doi: 10.1371/journal.pone.0247093

Gene expression profiling identifies FLT3 mutation-like cases in wild-type FLT3 acute myeloid leukemia

Adrián Mosquera Orgueira ^1,^2,^3,^*, Andrés Peleteiro Raíndo ^1,^2,^3,^‡, Miguel Cid López ^1,^2,^3,^‡, Beatriz Antelo Rodríguez ^1,^2,³, José Ángel Díaz Arias ^1,², Roi Ferreiro Ferro ^1,², Natalia Alonso Vence ^1,², Ángeles Bendaña López ^1,², Aitor Abuín Blanco ^1,², Laura Bao Pérez ^1,², Paula Melero Valentín ¹, Marta Sonia González Pérez ^1,², Claudio Cerchione ⁴, Giovanni Martinelli ⁵, Pau Montesinos Fernández ⁶, Manuel Mateo Pérez Encinas ^1,^2,³, José Luis Bello López ^1,^2,³

Editor: Francesco Bertolini⁷

¹Health Research Institute of Santiago de Compostela (IDIS), Santiago, Spain

²Division of Hematology, Complexo Hospitalario Universitario de Santiago de Compostela (CHUS), SERGAS, Santiago, Spain

³University of Santiago de Compostela, Santiago, Spain

⁴University of Bologna, Bologna, Italy

⁵Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori, Meldola, Italy

⁶University Hospital of La Fe in Valencia, Valencia, Spain

⁷European Institute of Oncology, ITALY

Competing Interests: The authors have declared that no competing interests exist.

‡ These authors contributed equally as second coauthors on this work.

^✉

* E-mail: adrian.mosquera@live.com

Roles

Adrián Mosquera Orgueira: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Resources, Visualization, Writing – original draft

Andrés Peleteiro Raíndo: Conceptualization, Investigation, Writing – original draft

Miguel Cid López: Conceptualization, Investigation, Methodology, Writing – original draft

Beatriz Antelo Rodríguez: Project administration, Visualization, Writing – original draft

José Ángel Díaz Arias: Conceptualization, Investigation, Methodology, Resources, Writing – original draft

Roi Ferreiro Ferro: Investigation, Methodology, Visualization, Writing – original draft

Natalia Alonso Vence: Writing – original draft

Ángeles Bendaña López: Writing – original draft, Writing – review & editing

Aitor Abuín Blanco: Writing – original draft

Laura Bao Pérez: Investigation, Writing – original draft

Paula Melero Valentín: Conceptualization, Writing – original draft

Marta Sonia González Pérez: Writing – original draft, Writing – review & editing

Claudio Cerchione: Writing – review & editing

Giovanni Martinelli: Writing – review & editing

Pau Montesinos Fernández: Writing – review & editing

Manuel Mateo Pérez Encinas: Writing – review & editing

José Luis Bello López: Writing – review & editing

Francesco Bertolini: Editor

PMCID: PMC7886212 PMID: 33592069

Abstract

Background

FLT3 mutation is present in 25–30% of all acute myeloid leukemias (AML), and it is associated with adverse outcome. FLT3 inhibitors have shown improved survival results in AML both as upfront treatment and in relapsed/refractory disease. Curiously, a variable proportion of wild-type FLT3 patients also responded to these drugs.

Methods

We analyzed 6 different transcriptomic datasets of AML cases. Differential expression between mutated and wild-type FLT3 AMLs was performed with the Wilcoxon-rank sum test. Hierarchical clustering was used to identify FLT3-mutation like AMLs. Finally, enrichment in recurrent mutations was performed with the Fisher’s test.

Results

A FLT3 mutation-like gene expression pattern was identified among wild-type FLT3 AMLs. This pattern was highly enriched in NPM1 and DNMT3A mutants, and particularly in combined NPM1/DNMT3A mutants.

Conclusions

We identified a FLT3 mutation-like gene expression pattern in AML which was highly enriched in NPM1 and DNMT3A mutations. Future analysis about the predictive role of this biomarker among wild-type FLT3 patients treated with FLT3 inhibitors is envisaged.

1. Introduction

FMS-like tyrosine kinase-3 (FLT3) is a receptor tyrosine kinase commonly mutated in acute myeloid leukemia (AML) [1]. FLT3 internal tandem duplication (ITD) is the most common mutation, affecting 25% of de novo AML cases [2], and additional tyrosine kinase mutations (FLT3-TKD) in codons D385 and I386 have been observed in 5–10% of cases [3]. Both types of mutations induce constitutive activation of FLT3 kinase activity, inducing pro-survival and proliferative signals [4]. FLT3-ITD mutation confers a poor outcome in de novo and relapsed AML, and it is currently suggested to include these patients in clinical trials [5, 6].

A number of promising FLT3 inhibitors are under development for AML treatment. The addition of the multi-kinase inhibitor midostaurin [7] to standard chemotherapy in newly diagnosed FLT3-mutated (FLT3^mut) AML significantly improved event-free survival and overall survival in all FLT3 mutation subtypes [8]. Several novel FLT3 inhibitors with increased specificity for FLT3 are under study, such as gilteritinib, crenolanib and quizartinib [3], with encouraging results from phase 2 and 3 trials in the relapsed & refractory AML setting [9–11]. Curiously, overall responses among wild-type FLT3 AML patients were reported for both gilteritinib (12% of composite overall responses) and quizartinib (30–36% of composite complete remissions) [9, 11]. It has been hypothesized that this effect could be due to nonspecific inhibition of other kinases, cryptic FLT3 activation by other mutations or hyperactivation of FLT3 by its ligand [9]. Not surprisingly, 2 clinical trials are currently underway in order to study the activity of FLT3 inhibitors in wild-type FLT3 patients [12, 13], but there is still no biomarker to predict which patients will respond to these drugs. Therefore, an interesting point would be to identify gene expression changes associated with FLT3 mutation in order to search for wild-type FLT3 cases that resemble FLT3^mut AMLs at the transcriptomic level.

In this study we analyzed the transcriptomic pattern of 6 different AML cohorts, which enabled the identification of a FLT3 mutation-like gene expression pattern highly enriched in NPM1 and DNMT3A mutants. Our results indicate common deregulated pathways among these leukemias, opening the way to study the role of this biomarker in order to predict responses to FLT3 inhibitors in wild-type FLT3 AML.

2. Materials and methods

We analyzed five AML gene expression datasets available in the Gene Expression Omnibus (GEO): GSE14468 (461 cases of de novo cytogenetically normal AML), GSE10358 (188 cases of de novo AML), GSE61804 (279 de novo AML cases), GSE17855 (237 pediatric AML cases) and GSE15434 (251 cases of normal karyotype AML). In the case of GSE14468 and GSE10358, both FLT3-ITD and D385 mutation status were reported, whereas in the remaining datasets only the FLT3-ITD status was informed. All datasets used the same type of gene expression arrays: Affymetrix Human Genome U133 Plus 2.0 Array. Signal intensities were rank-normalized, and differential expression between FLT3^mut (either ITD or TKD) and wild-type samples was performed in the largest cohort (GSE14468) using the two-sided Wilcoxon-rank sum test. P-values were adjusted for multiple testing using the Bonferroni method. Hierarchical clusterization and heatmap plots were created using the heatmaps3 package with default parameters [14], and enrichment analysis was performed with the two-sided exact Fisher’s test. Gene ontology analysis was performed on WebGestalt with default parameters (FDR significance threshold, 0.05) [15].

RNAseq and mutation data from 246 AML patients treated with intensive chemotherapy was retrieved from Bamopoulos et al. (GEO identification GSE146173) [16]. Raw count data was transformed to normalized transcripts per million using the fpkm function implemented in the DESEQ2 package [17]. Afterwards, rank-transformation was applied. 595 genes matching genes with the FLT3-like pattern were selected, followed by standard hierarchical clusterization. Differential mutation between the FLT3-like and no-FLT3-like group was performed with Fisher’s test.

All computations except gene ontology analysis were performed in R. All data used for this analysis is readily accessible from public repositories.

3. Results

FLT3-like gene expression pattern

We chose the largest database (GSE14468) as the discovery set, and we identified 911 probes mapping to 649 different genes which were differentially expressed between FLT3^mut (ITD and/or TKD) and wild-type FLT3 samples (Bonferroni p-value <0.05, S1 Table). None of the probes mapped to FLT3. These probes were significantly enriched in 52 gene ontology terms, among which 17 terms were associated with hemopoiesis & immunology, and 3 terms were specifically linked to myeloid differentiation, namely myeloid cell homeostasis, q-value 5.01 x 10⁻³; myeloid cell differentiation, q-value 4.86 x 10⁻³; and negative regulation of myeloid cell differentiation, q-value 0.03 (S2 Table). Hierarchical clusterization revealed two broad groups. A cluster of 46.20% of patients was substantially enriched in FLT3 mutants (p-value < 1 x 10⁻⁴), since it contained 81.67% of all FLT3-ITD cases, 61.36% of all FLT3-TKD cases and 83.33 of composed mutants (FLT3-ITD plus FLT3-TKD; Fig 1A). 28.52% of wild-type FLT3 cases were also clustered within this group. A similar finding was identified in the independent GSE10358 database, where a cluster of 51.06% of patients contained 81.08% of all FLT3-ITD mutants and 70.00% of all FLT3-TKD mutants (p-value < 1 x 10⁻⁴). Furthermore, 42.15% of wild-type FLT3 cases were also clustered within this group (Fig 1B).

Fig 1 — **A and B)** Heatmaps representing the hierarchical clustering of AML cases according to the expression of the 911 probes associated with *FLT3* ITD and TKD mutation status. The color coding of the bar indicates if a patient harbos a *FLT3*-ITD mutation (red bars), a *FLT3*-TKD mutation (green bars), both mutations (yellow bars) or no *FLT3* mutation (light blue bars). **C-E)** Heatmaps representing the hierarchical clustering of AML cases according to the expression of the 911 probes associated with *FLT3*-ITD mutation status. No information about *FLT3*-TKD mutations was reported for patients in these databases. Red bars indicate *FLT3*-ITD cases and light blue bars indicate lack of *FLT3*-ITD mutation.

The same clustering was repeated in 3 different datasets that only reported FLT3-ITD mutation status. In GSE61804 a cluster comprising 58.06% of all patients harbored 78% of all FLT3-ITD cases (p-value 1.50 x 10⁻³), and 53.71% of all non FLT3-ITD cases were grouped within this cluster (Fig 1C). In GSE17855, a cluster of 47.67% of patients contained 81.25% of all FLT3-ITD patients (p-value < 1 x 10⁻⁴), but it also included 39.15% of all non FLT3-ITD cases (Fig 1D). Finally, in GSE15434, a cluster of 64.14% of patients was enriched in FLT3-ITD cases, comprising 86.67% of the whole cohort (p-value < 1 x 10⁻⁴); and additionally 49.11% of all non FLT3-ITD cases co-clustered within this group (Fig 1E). We replicated the FLT3-like pattern in GSE146173 using RNAseq data, identifying 28.49% of wild-type FLT3 AMLs as FLT3-like (Fig 2).

Fig 2 — The color coding of the bar follows the same pattern as that of Fig 1.

Mutation landscape of FLT3-like leukemias

Four of the microarray databases provided data about driver mutations in a few genes (S3 Table). The most significant finding was the enrichment of FLT3-like AMLs in NPM1 mutants, which was observed in the 4 cohorts. This proportion was variable, ranging from 80.72% in karyotype normal AML (GSE15434) to just 12.16% in pediatric AML (GSE17855), indicating that the existence of the FLT3-like pattern is not explained by co-occurring NPM1 mutations. Additionally, a relative enrichment in IDH1 mutants within FLT3-like leukemias was detected in the discovery set GSE14468 (p-value 2.07 x 10⁻⁴). Furthermore, depletion of CEBPA mutants in FLT3-like AML was a common phenomenon in all 3 cohorts that reported mutations in this gene, and a reduced frequency of KIT mutations was also discovered in the pediatric AML dataset (GSE17855).

Using the most recent RNAseq data published by Bamopoulos et al. [16] we observed that FLT3-like leukemias were highly enriched in concurrent NPM1 and DNMT3A mutants, whereas 19.60% of FLT3-like AMLs were NPM1 and DNMT3A wild-type. Additionally, there was a tendency for an enrichment in IDH1 mutations (p-value 0.09). On the contrary, FLT3-like AMLs were depleted in ASXL1, CEBPA, RUNX1, TP53, SF3B1 and U2AF1 mutations (Table 1).

Table 1. Differential distribution of recurrently mutated genes in wild-type FLT3 AML patients according to the presence or absence of the FLT3-like pattern (GSE146173).

Gene ID	p-value	FLT3-like (%)	No FLT3-like (%)
ASXL1	5.29E-04	1.96	21.87
CEBPA	2.66E-15	0	5.47
DNMT3A	1.61E-03	41.17	17.19
EZH2	1	1.96	1.56
IDH1	0.09	15.69	7.03
IDH2	0.81	13.73	12.5
NPM1	3.40E-24	74.51	2.34
RUNX1	2.30E-05	1.96	28.12
TET2	0.29	23.53	16.41
TP53	0.02	1.96	14.06
SRSF2	0.62	9.8	13.28
U2AF1	7.95E-14	0	7.03
SF3B1	8.10E-18	0	3.12
Concurrent NPM1 & DNMT3A	7.11E-05	19.61	1.56

Open in a new tab

4. Discussion

In this report we describe the existence of a FLT3 mutation-like gene expression pattern in wild-type FLT3 AMLs. The FLT3-mutation like pattern was enriched in NPM1 and DNMT3A mutant leukemias. This is probably related to the significant co-occurrence of FLT3 mutations with those of NPM1 and DNMT3A [18], which probably leads to partially overlapping transcriptomic fingerprints. Not surprisingly, the FLT3-like pattern contains numerous HOX genes, which have been previously vinculated with the NPM1-transcriptional pattern [19]. Nevertheless, the heterogeneous frequency of NPM1 mutations among FLT3-like leukemias, and particularly its low frequency in pediatric AML, along with the reproducibility of the FLT3-like pattern in all cohorts, indicate that co-occurring NPM1 mutations are insufficient to explain the existence of the FLT3-like pattern.

Our results suggest that a group of AMLs with FLT3 plus NPM1 and/or DNMT3A mutations share a similar transcriptomic background. Noteworthy, responses to the FLT3 inhibitor gilteritinib among relapsed FLT3^mut AMLs are superior in those patients with mutations in NPM1 or DNMT3A, and particularly in those with both genes mutated [20], and similar findings were reported with crenolanib [21]. Furthermore, FLT3-ITD leukemias with mutations in NPM1 or DNMT3A exhibit a different drug response mechanism to the FLT3 inhibitor quizartinib, where the cell differentiation effect predominates over the cytotoxic mechanism [22].

The use of FLT3 inhibitors in wild-type FLT3 AML has been tested in some trials. For example, the FLT3 inhibitor midostaurin evidenced blast reduction responses in 53% of relapsed & refractory AML cases [23]. Similarly, responses to novel FLT3 inhibitors in a variable proportion of wild-type FLT3 cases have been observed in phase I & II trials [24–26]. This has motivated the development of new studies to specifically test the possible benefit of adding these drugs in the upfront treatment for AML [27, 28]. As it is expected that only a subgroup of patients might benefit from FLT3 inhibitors, it is imperative to develop and test new predictive biomarkers of response. Therefore, it becomes necessary to test the predictive value of the FLT3-like pattern in these clinical trials. Additionally, the enrichment of FLT3-like leukemias in IDH1 mutations (which are correlated with NPM1 mutation [29]) could set the basis for the development of new clinical trials testing the combination of different check-point inhibitors in AML [30].

This study has some limitations. Firstly, a variable proportion of FLT3 mutation-like samples were clustered near FLT3 mutants across the different cohorts, which probably reflects substantial heterogeneity between them. One of the possible explanations for this could be a differential distribution of NPM1 and DNMT3A mutants, since these are drivers of cytogenetically-normal AMLs (such as in the case of GSE15434) [31]. Finally, some of the datasets had only FLT3-ITD annotation, and a minority of the FLT3-like cases might indeed have FLT3-TKD mutations.

5. Conclusions

Our results are concordant with the existence of a FLT3 mutation-like transcriptomic pattern with a different mutational background which clusters a proportion of wild-type FLT3 AMLs with FLT3^mut samples. These leukemias were highly enriched in NPM1, and particularly in composed NPM1/DNMT3A mutants, but NPM1 status alone was insufficient to explain the existence of the FLT3-like pattern. The analysis of wild-type FLT3 AML patients treated with FLT3 inhibitors in clinical trials is envisaged in order to study its possible role as a drug response biomarker.

Supporting information

S1 Table. Annotation of all probes significantly associated with FLT3 mutation status in AML.

(XLSX)

Click here for additional data file.^{(37.4KB, xlsx)}

S2 Table. Significantly enriched gene ontology (biological process) terms from the list of genes associated with FLT3 mutation status.

(XLSX)

Click here for additional data file.^{(32.1KB, xlsx)}

S3 Table. Differential distribution of recurrently mutated genes in wild-type FLT3 AML wild-type according to the presence or absence of the FLT3-like pattern.

Data reported for mutations analyzed in GSE14468, GSE10358, GSE15434 and GSE17855.

(XLSX)

Click here for additional data file.^{(9.4KB, xlsx)}

Acknowledgments

We’d like to thank the Supercomputing Center of Galicia (CESGA) for their contribution.

All authors approved the final version of this manuscript.

Data Availability

The data is publicly available in Gene Expression Omnibus with the following ids: GSE14468, GSE10358, GSE61804, GSE17855, GSE15434 and GSE146173.

Funding Statement

The author(s) received no specific funding for this work.

References

1.Levis M, Small D. FLT3: ITDoes matter in leukemia. Leukemia. 2003. September;17(9):1738–52. Review. 10.1038/sj.leu.2403099 . [DOI] [PubMed] [Google Scholar]
2.Stirewalt DL, Radich JP. The role of FLT3 in haematopoietic malignancies. Nat Rev Cancer. 2003. September;3(9):650–65. Review. 10.1038/nrc1169 . [DOI] [PubMed] [Google Scholar]
3.Daver N, Schlenk RF, Russell NH, Levis MJ. Targeting FLT3 mutations in AML: review of current knowledge and evidence. Leukemia. 2019. February;33(2):299–312. Epub 2019 Jan 16. Review. 10.1038/s41375-018-0357-9 . [DOI] [PMC free article] [PubMed] [Google Scholar]
4.Grafone T, Palmisano M, Nicci C, Storti S. An overview on the role of FLT3-tyrosine kinase receptor in acute myeloid leukemia: biology and treatment. Oncol Rev. 2012. April 17;6(1):e8 eCollection 2012 Mar 5. Review. 10.4081/oncol.2012.e8 . [DOI] [PMC free article] [PubMed] [Google Scholar]
5.O’Donnell MR, Tallman MS, Abboud CN, Altman JK, Appelbaum FR, Arber DA, et al. Acute Myeloid Leukemia, Version 3.2017, NCCN Clinical Practice Guidelines in Oncology. J Natl Compr Canc Netw. 2017. July;15(7):926–957. 10.6004/jnccn.2017.0116 . [DOI] [PubMed] [Google Scholar]
6.Papaemmanuil E, Gerstung M, Bullinger L, Gaidzik VI, Paschka P, Roberts ND, et al. Genomic Classification and Prognosis in Acute Myeloid Leukemia. N Engl J Med. 2016. June 9;374(23):2209–2221. 10.1056/NEJMoa1516192 . [DOI] [PMC free article] [PubMed] [Google Scholar]
7.Stone RM, Mandrekar SJ, Sanford BL, et al. Midostaurin plus Chemotherapy for Acute Myeloid Leukemia with a FLT3 Mutation. N Engl J Med. 2017;377(5):454–464. 10.1056/NEJMoa1614359 [DOI] [PMC free article] [PubMed] [Google Scholar]
8.Stone RM, Mandrekar SJ, Sanford BL, Laumann K, Geyer SM, Bloomfield CD, et al. The Addition of Midostaurin to Standard Chemotherapy Decreases Cumulative Incidence of Relapse (CIR) in the International Prospective Randomized, Placebo-Controlled, Double-Blind Trial (CALGB 10603 / RATIFY [Alliance]) for Newly Diagnosed Acute Myeloid Leukemia (AML) Patients with FLT3 Mutations. Blood (2017) 130 (Supplement 1): 2580 10.1182/blood.V130.Suppl_1.2580.2580 [DOI] [Google Scholar]
9.Perl AE, Martinelli G, Cortes JE, et al. Gilteritinib or Chemotherapy for Relapsed or Refractory FLT3-Mutated AML. N Engl J Med. 2019;381(18):1728–1740. 10.1056/NEJMoa1902688 [DOI] [PubMed] [Google Scholar]
10.Randhawa JK, Kantarjian HM, Borthakur G, Thompson PA, Konopleva M, Daver N, et al. Results of a Phase II Study of Crenolanib in Relapsed/Refractory Acute Myeloid Leukemia Patients (Pts) with Activating FLT3 Mutations. Blood (2014) 124 (21): 389 10.1182/blood.V124.21.389.389 [DOI] [Google Scholar]
11.Cortes JE, Khaled S, Martinelli G, et al. Quizartinib versus salvage chemotherapy in relapsed or refractory FLT3-ITD acute myeloid leukaemia (QuANTUM-R): a multicentre, randomised, controlled, open-label, phase 3 trial [published correction appears in Lancet Oncol. 2019 Jul;20(7):e346]. Lancet Oncol. 2019;20(7):984–997. 10.1016/S1470-2045(19)30150-0 [DOI] [PubMed] [Google Scholar]
12.ClinicalTrials.gov [Internet]. Bethesda (MD): National Library of Medicine (US). 2000 Feb 29 -. Identifier NCT04107727 Trial to Compare Efficacy and Safety of Chemotherapy/Quizartinib vs Chemotherapy/Placebo in Adults FMS-like Tyrosine Kinase 3 (FLT3) Wild-type Acute Myeloid Leukemia (AML); 2017 Sept 27 [cited 2020 Feb 27]; [about 4 screens]. https://clinicaltrials.gov/ct2/show/NCT04107727
13.ClinicalTrials.gov [Internet]. Bethesda (MD): National Library of Medicine (US). 2000 Feb 29 -. Identifier NCT03512197A Global Study of the Efficacy and Safety of Midostaurin + Chemotherapy in Newly Diagnosed Patients With FLT3 Mutation Negative (FLT3-MN) Acute Myeloid Leukemia (AML); 2019 Apr 30 [cited 2020 Feb 27]; [about 4 screens]. https://clinicaltrials.gov/ct2/show/NCT03512197
14.Zhao S, Guo Y, Sheng Q, Shyr Y. Advanced heat map and clustering analysis using heatmap3. Biomed Res Int. 2014;2014:986048 Epub 2014 Jul 16. 10.1155/2014/986048 . [DOI] [PMC free article] [PubMed] [Google Scholar]
15.Liao Y, Wang J, Jaehnig EJ, Shi Z, Zhang B. WebGestalt 2019: gene set analysis toolkit with revamped UIs and APIs. Nucleic Acids Res. 2019. July 2;47(W1):W199–W205. 10.1093/nar/gkz401 . [DOI] [PMC free article] [PubMed] [Google Scholar]
16.Bamopoulos SA, Batcha AMN, Jurinovic V, et al. Clinical presentation and differential splicing of SRSF2, U2AF1 and SF3B1 mutations in patients with acute myeloid leukemia [published online ahead of print, 2020 May 1]. Leukemia. 2020; 10.1038/s41375-020-0839-4 [DOI] [PubMed] [Google Scholar]
17.Love MI, Huber W, Anders S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 2014;15(12):550 10.1186/s13059-014-0550-8 [DOI] [PMC free article] [PubMed] [Google Scholar]
18.Bezerra MF, Lima AS, Piqué-Borràs MR, et al. Co-occurrence of DNMT3A, NPM1, FLT3 mutations identifies a subset of acute myeloid leukemia with adverse prognosis. Blood. 2020;135(11):870–875. 10.1182/blood.2019003339 [DOI] [PubMed] [Google Scholar]
19.Mullighan CG, Kennedy A, Zhou X, et al. Pediatric acute myeloid leukemia with NPM1 mutations is characterized by a gene expression profile with dysregulated HOX gene expression distinct from MLL-rearranged leukemias. Leukemia. 2007;21(9):2000–2009. 10.1038/sj.leu.2404808 [DOI] [PubMed] [Google Scholar]
20.Levis MJ, Perl AE, Martinelli G et al. Effect of gilteritinib on survival in patients with FLT3-mutated (FLT3mut+) relapsed/refractory (R/R) AML who have common AML co-mutations or a high FLT3-ITD allelic ratio. Journal of Clinical Oncology 2019. 37:15_suppl, 7000–7000 [Google Scholar]
21.Goldberg AD, Collins RH, Stone RM et al. Addition of Crenolanib to Induction Chemotherapy Overcomes the Poor Prognostic Impact of Co- Occurring Driver Mutations in Patients with Newly Diagnosed FLT3-Mutated AML. Blood (2018) 132 (Supplement 1): 1436 10.1182/blood-2018-99-117016 [DOI] [Google Scholar]
22.Nybakken GE, Canaani J, Roy D, et al. Quizartinib elicits differential responses that correlate with karyotype and genotype of the leukemic clone. Leukemia. 2016;30(6):1422–1425. 10.1038/leu.2015.320 [DOI] [PMC free article] [PubMed] [Google Scholar]
23.Fischer T, Stone RM, Deangelo DJ, et al. Phase IIB trial of oral Midostaurin (PKC412), the FMS-like tyrosine kinase 3 receptor (FLT3) and multi-targeted kinase inhibitor, in patients with acute myeloid leukemia and high-risk myelodysplastic syndrome with either wild-type or mutated FLT3. J Clin Oncol. 2010;28(28):4339–4345. 10.1200/JCO.2010.28.9678 [DOI] [PMC free article] [PubMed] [Google Scholar]
24.Perl AE, Altman JK, Cortes J, et al. Selective inhibition of FLT3 by gilteritinib in relapsed or refractory acute myeloid leukaemia: a multicentre, first-in-human, open-label, phase 1–2 study. Lancet Oncol. 2017. August;18(8):1061–1075. Epub 2017 Jun 20. Erratum in: Lancet Oncol. 2017 Dec;18(12): e711. Lancet Oncol. 2018 Jul;19(7):e335. Lancet Oncol. 2019 Jun;20(6):e293. 10.1016/S1470-2045(17)30416-3 . [DOI] [PMC free article] [PubMed] [Google Scholar]
25.Randhawa JK, Kantarjian HM, Borthakur G et al. Results of a Phase II Study of Crenolanib in Relapsed/Refractory Acute Myeloid Leukemia Patients (Pts) with Activating FLT3 Mutations. Blood (2014) 124 (21): 389 10.1182/blood.V124.21.389.389 [DOI] [Google Scholar]
26.Cortes J, Perl AE, Döhner H, et al. Quizartinib, an FLT3 inhibitor, as monotherapy in patients with relapsed or refractory acute myeloid leukaemia: an open-label, multicentre, single-arm, phase 2 trial. Lancet Oncol. 2018. July;19(7):889–903. Epub 2018 May 31. 10.1016/S1470-2045(18)30240-7 . [DOI] [PMC free article] [PubMed] [Google Scholar]
27.ClinicalTrials.gov [Internet]. Bethesda (MD): National Library of Medicine (US). 2000 Feb 29 -. Identifier NCT04107727 Trial to Compare Efficacy and Safety of Chemotherapy/Quizartinib vs Chemotherapy/Placebo in Adults FMS-like Tyrosine Kinase 3 (FLT3) Wild-type Acute Myeloid Leukemia (AML); 2017 Sept 27 [cited 2020 Feb 27]; [about 4 screens]. https://clinicaltrials.gov/ct2/show/NCT04107727
28.ClinicalTrials.gov [Internet]. Bethesda (MD): National Library of Medicine (US). 2000 Feb 29 -. Identifier NCT03512197A Global Study of the Efficacy and Safety of Midostaurin + Chemotherapy in Newly Diagnosed Patients With FLT3 Mutation Negative (FLT3-MN) Acute Myeloid Leukemia (AML); 2019 Apr 30 [cited 2020 Feb 27]; [about 4 screens]. https://clinicaltrials.gov/ct2/show/NCT03512197
29.Falini B, Spinelli O, Meggendorfer M, et al. IDH1-R132 changes vary according to NPM1 and other mutations status in AML. Leukemia. 2019;33(4):1043–1047. 10.1038/s41375-018-0299-2 [DOI] [PMC free article] [PubMed] [Google Scholar]
30.DiNardo CD, Stein EM, de Botton S, et al. Durable Remissions with Ivosidenib in IDH1-Mutated Relapsed or Refractory AML. N Engl J Med. 2018;378(25):2386–2398. 10.1056/NEJMoa1716984 [DOI] [PubMed] [Google Scholar]
31.Wang M, Yang C, Zhang L, Schaar DG. Molecular Mutations and Their Cooccurrences in Cytogenetically Normal Acute Myeloid Leukemia. Stem Cells Int. 2017;2017:6962379 10.1155/2017/6962379 [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 Table. Annotation of all probes significantly associated with FLT3 mutation status in AML.

(XLSX)

Click here for additional data file.^{(37.4KB, xlsx)}

S2 Table. Significantly enriched gene ontology (biological process) terms from the list of genes associated with FLT3 mutation status.

(XLSX)

Click here for additional data file.^{(32.1KB, xlsx)}

S3 Table. Differential distribution of recurrently mutated genes in wild-type FLT3 AML wild-type according to the presence or absence of the FLT3-like pattern.

Data reported for mutations analyzed in GSE14468, GSE10358, GSE15434 and GSE17855.

(XLSX)

Click here for additional data file.^{(9.4KB, xlsx)}

Data Availability Statement

The data is publicly available in Gene Expression Omnibus with the following ids: GSE14468, GSE10358, GSE61804, GSE17855, GSE15434 and GSE146173.

[pone.0247093.ref001] 1.Levis M, Small D. FLT3: ITDoes matter in leukemia. Leukemia. 2003. September;17(9):1738–52. Review. 10.1038/sj.leu.2403099 . [DOI] [PubMed] [Google Scholar]

[pone.0247093.ref002] 2.Stirewalt DL, Radich JP. The role of FLT3 in haematopoietic malignancies. Nat Rev Cancer. 2003. September;3(9):650–65. Review. 10.1038/nrc1169 . [DOI] [PubMed] [Google Scholar]

[pone.0247093.ref003] 3.Daver N, Schlenk RF, Russell NH, Levis MJ. Targeting FLT3 mutations in AML: review of current knowledge and evidence. Leukemia. 2019. February;33(2):299–312. Epub 2019 Jan 16. Review. 10.1038/s41375-018-0357-9 . [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0247093.ref004] 4.Grafone T, Palmisano M, Nicci C, Storti S. An overview on the role of FLT3-tyrosine kinase receptor in acute myeloid leukemia: biology and treatment. Oncol Rev. 2012. April 17;6(1):e8 eCollection 2012 Mar 5. Review. 10.4081/oncol.2012.e8 . [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0247093.ref005] 5.O’Donnell MR, Tallman MS, Abboud CN, Altman JK, Appelbaum FR, Arber DA, et al. Acute Myeloid Leukemia, Version 3.2017, NCCN Clinical Practice Guidelines in Oncology. J Natl Compr Canc Netw. 2017. July;15(7):926–957. 10.6004/jnccn.2017.0116 . [DOI] [PubMed] [Google Scholar]

[pone.0247093.ref006] 6.Papaemmanuil E, Gerstung M, Bullinger L, Gaidzik VI, Paschka P, Roberts ND, et al. Genomic Classification and Prognosis in Acute Myeloid Leukemia. N Engl J Med. 2016. June 9;374(23):2209–2221. 10.1056/NEJMoa1516192 . [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0247093.ref007] 7.Stone RM, Mandrekar SJ, Sanford BL, et al. Midostaurin plus Chemotherapy for Acute Myeloid Leukemia with a FLT3 Mutation. N Engl J Med. 2017;377(5):454–464. 10.1056/NEJMoa1614359 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0247093.ref008] 8.Stone RM, Mandrekar SJ, Sanford BL, Laumann K, Geyer SM, Bloomfield CD, et al. The Addition of Midostaurin to Standard Chemotherapy Decreases Cumulative Incidence of Relapse (CIR) in the International Prospective Randomized, Placebo-Controlled, Double-Blind Trial (CALGB 10603 / RATIFY [Alliance]) for Newly Diagnosed Acute Myeloid Leukemia (AML) Patients with FLT3 Mutations. Blood (2017) 130 (Supplement 1): 2580 10.1182/blood.V130.Suppl_1.2580.2580 [DOI] [Google Scholar]

[pone.0247093.ref009] 9.Perl AE, Martinelli G, Cortes JE, et al. Gilteritinib or Chemotherapy for Relapsed or Refractory FLT3-Mutated AML. N Engl J Med. 2019;381(18):1728–1740. 10.1056/NEJMoa1902688 [DOI] [PubMed] [Google Scholar]

[pone.0247093.ref010] 10.Randhawa JK, Kantarjian HM, Borthakur G, Thompson PA, Konopleva M, Daver N, et al. Results of a Phase II Study of Crenolanib in Relapsed/Refractory Acute Myeloid Leukemia Patients (Pts) with Activating FLT3 Mutations. Blood (2014) 124 (21): 389 10.1182/blood.V124.21.389.389 [DOI] [Google Scholar]

[pone.0247093.ref011] 11.Cortes JE, Khaled S, Martinelli G, et al. Quizartinib versus salvage chemotherapy in relapsed or refractory FLT3-ITD acute myeloid leukaemia (QuANTUM-R): a multicentre, randomised, controlled, open-label, phase 3 trial [published correction appears in Lancet Oncol. 2019 Jul;20(7):e346]. Lancet Oncol. 2019;20(7):984–997. 10.1016/S1470-2045(19)30150-0 [DOI] [PubMed] [Google Scholar]

[pone.0247093.ref012] 12.ClinicalTrials.gov [Internet]. Bethesda (MD): National Library of Medicine (US). 2000 Feb 29 -. Identifier NCT04107727 Trial to Compare Efficacy and Safety of Chemotherapy/Quizartinib vs Chemotherapy/Placebo in Adults FMS-like Tyrosine Kinase 3 (FLT3) Wild-type Acute Myeloid Leukemia (AML); 2017 Sept 27 [cited 2020 Feb 27]; [about 4 screens]. https://clinicaltrials.gov/ct2/show/NCT04107727

[pone.0247093.ref013] 13.ClinicalTrials.gov [Internet]. Bethesda (MD): National Library of Medicine (US). 2000 Feb 29 -. Identifier NCT03512197A Global Study of the Efficacy and Safety of Midostaurin + Chemotherapy in Newly Diagnosed Patients With FLT3 Mutation Negative (FLT3-MN) Acute Myeloid Leukemia (AML); 2019 Apr 30 [cited 2020 Feb 27]; [about 4 screens]. https://clinicaltrials.gov/ct2/show/NCT03512197

[pone.0247093.ref014] 14.Zhao S, Guo Y, Sheng Q, Shyr Y. Advanced heat map and clustering analysis using heatmap3. Biomed Res Int. 2014;2014:986048 Epub 2014 Jul 16. 10.1155/2014/986048 . [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0247093.ref015] 15.Liao Y, Wang J, Jaehnig EJ, Shi Z, Zhang B. WebGestalt 2019: gene set analysis toolkit with revamped UIs and APIs. Nucleic Acids Res. 2019. July 2;47(W1):W199–W205. 10.1093/nar/gkz401 . [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0247093.ref016] 16.Bamopoulos SA, Batcha AMN, Jurinovic V, et al. Clinical presentation and differential splicing of SRSF2, U2AF1 and SF3B1 mutations in patients with acute myeloid leukemia [published online ahead of print, 2020 May 1]. Leukemia. 2020; 10.1038/s41375-020-0839-4 [DOI] [PubMed] [Google Scholar]

[pone.0247093.ref017] 17.Love MI, Huber W, Anders S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 2014;15(12):550 10.1186/s13059-014-0550-8 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0247093.ref018] 18.Bezerra MF, Lima AS, Piqué-Borràs MR, et al. Co-occurrence of DNMT3A, NPM1, FLT3 mutations identifies a subset of acute myeloid leukemia with adverse prognosis. Blood. 2020;135(11):870–875. 10.1182/blood.2019003339 [DOI] [PubMed] [Google Scholar]

[pone.0247093.ref019] 19.Mullighan CG, Kennedy A, Zhou X, et al. Pediatric acute myeloid leukemia with NPM1 mutations is characterized by a gene expression profile with dysregulated HOX gene expression distinct from MLL-rearranged leukemias. Leukemia. 2007;21(9):2000–2009. 10.1038/sj.leu.2404808 [DOI] [PubMed] [Google Scholar]

[pone.0247093.ref020] 20.Levis MJ, Perl AE, Martinelli G et al. Effect of gilteritinib on survival in patients with FLT3-mutated (FLT3mut+) relapsed/refractory (R/R) AML who have common AML co-mutations or a high FLT3-ITD allelic ratio. Journal of Clinical Oncology 2019. 37:15_suppl, 7000–7000 [Google Scholar]

[pone.0247093.ref021] 21.Goldberg AD, Collins RH, Stone RM et al. Addition of Crenolanib to Induction Chemotherapy Overcomes the Poor Prognostic Impact of Co- Occurring Driver Mutations in Patients with Newly Diagnosed FLT3-Mutated AML. Blood (2018) 132 (Supplement 1): 1436 10.1182/blood-2018-99-117016 [DOI] [Google Scholar]

[pone.0247093.ref022] 22.Nybakken GE, Canaani J, Roy D, et al. Quizartinib elicits differential responses that correlate with karyotype and genotype of the leukemic clone. Leukemia. 2016;30(6):1422–1425. 10.1038/leu.2015.320 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0247093.ref023] 23.Fischer T, Stone RM, Deangelo DJ, et al. Phase IIB trial of oral Midostaurin (PKC412), the FMS-like tyrosine kinase 3 receptor (FLT3) and multi-targeted kinase inhibitor, in patients with acute myeloid leukemia and high-risk myelodysplastic syndrome with either wild-type or mutated FLT3. J Clin Oncol. 2010;28(28):4339–4345. 10.1200/JCO.2010.28.9678 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0247093.ref024] 24.Perl AE, Altman JK, Cortes J, et al. Selective inhibition of FLT3 by gilteritinib in relapsed or refractory acute myeloid leukaemia: a multicentre, first-in-human, open-label, phase 1–2 study. Lancet Oncol. 2017. August;18(8):1061–1075. Epub 2017 Jun 20. Erratum in: Lancet Oncol. 2017 Dec;18(12): e711. Lancet Oncol. 2018 Jul;19(7):e335. Lancet Oncol. 2019 Jun;20(6):e293. 10.1016/S1470-2045(17)30416-3 . [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0247093.ref025] 25.Randhawa JK, Kantarjian HM, Borthakur G et al. Results of a Phase II Study of Crenolanib in Relapsed/Refractory Acute Myeloid Leukemia Patients (Pts) with Activating FLT3 Mutations. Blood (2014) 124 (21): 389 10.1182/blood.V124.21.389.389 [DOI] [Google Scholar]

[pone.0247093.ref026] 26.Cortes J, Perl AE, Döhner H, et al. Quizartinib, an FLT3 inhibitor, as monotherapy in patients with relapsed or refractory acute myeloid leukaemia: an open-label, multicentre, single-arm, phase 2 trial. Lancet Oncol. 2018. July;19(7):889–903. Epub 2018 May 31. 10.1016/S1470-2045(18)30240-7 . [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0247093.ref027] 27.ClinicalTrials.gov [Internet]. Bethesda (MD): National Library of Medicine (US). 2000 Feb 29 -. Identifier NCT04107727 Trial to Compare Efficacy and Safety of Chemotherapy/Quizartinib vs Chemotherapy/Placebo in Adults FMS-like Tyrosine Kinase 3 (FLT3) Wild-type Acute Myeloid Leukemia (AML); 2017 Sept 27 [cited 2020 Feb 27]; [about 4 screens]. https://clinicaltrials.gov/ct2/show/NCT04107727

[pone.0247093.ref028] 28.ClinicalTrials.gov [Internet]. Bethesda (MD): National Library of Medicine (US). 2000 Feb 29 -. Identifier NCT03512197A Global Study of the Efficacy and Safety of Midostaurin + Chemotherapy in Newly Diagnosed Patients With FLT3 Mutation Negative (FLT3-MN) Acute Myeloid Leukemia (AML); 2019 Apr 30 [cited 2020 Feb 27]; [about 4 screens]. https://clinicaltrials.gov/ct2/show/NCT03512197

[pone.0247093.ref029] 29.Falini B, Spinelli O, Meggendorfer M, et al. IDH1-R132 changes vary according to NPM1 and other mutations status in AML. Leukemia. 2019;33(4):1043–1047. 10.1038/s41375-018-0299-2 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0247093.ref030] 30.DiNardo CD, Stein EM, de Botton S, et al. Durable Remissions with Ivosidenib in IDH1-Mutated Relapsed or Refractory AML. N Engl J Med. 2018;378(25):2386–2398. 10.1056/NEJMoa1716984 [DOI] [PubMed] [Google Scholar]

[pone.0247093.ref031] 31.Wang M, Yang C, Zhang L, Schaar DG. Molecular Mutations and Their Cooccurrences in Cytogenetically Normal Acute Myeloid Leukemia. Stem Cells Int. 2017;2017:6962379 10.1155/2017/6962379 [DOI] [PMC free article] [PubMed] [Google Scholar]

PERMALINK

Gene expression profiling identifies FLT3 mutation-like cases in wild-type FLT3 acute myeloid leukemia

Adrián Mosquera Orgueira

Andrés Peleteiro Raíndo

Miguel Cid López

Beatriz Antelo Rodríguez

José Ángel Díaz Arias

Roi Ferreiro Ferro

Natalia Alonso Vence

Ángeles Bendaña López

Aitor Abuín Blanco

Laura Bao Pérez

Paula Melero Valentín

Marta Sonia González Pérez

Claudio Cerchione

Giovanni Martinelli

Pau Montesinos Fernández

Manuel Mateo Pérez Encinas

José Luis Bello López

Roles

Abstract

Background

Methods

Results

Conclusions

1. Introduction

2. Materials and methods

3. Results

FLT3-like gene expression pattern

Fig 1.

Fig 2. Heatmaps representing the hierarchical clustering of AML cases according to the expression of the 595 matching genes in the RNAseq cohort (GSE146173).

Mutation landscape of FLT3-like leukemias

Table 1. Differential distribution of recurrently mutated genes in wild-type FLT3 AML patients according to the presence or absence of the FLT3-like pattern (GSE146173).

4. Discussion

5. Conclusions

Supporting information

Acknowledgments

Data Availability

Funding Statement

References

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

Similar articles

Cited by other articles

Links to NCBI Databases