Clinical characteristics and prognostic analysis of acute myeloid leukemia patients with PTPN11 mutations

ABSTRACT

Objectives

Little is known about the clinical impact of germline/somatic mutations of PTPN11 in acute leukemia. The aim of this study was to investigate the clinical characteristics and prognostic impact of PTPN11 mutations in patients with acute myeloid leukemia (AML).

Methods:

Seventy-four patients with PTPN11 mutation-positive AML treated at our institution were enrolled in this study. The prevalence of PTPN11 mutations was examined using targeted next-generation sequencing technology, and patients with AML and PTPN11 mutations were screened. Clinical characteristics, prognostic impact, and association between PTPN11 mutations and other mutations were analyzed retrospectively.

Results:

PTPN11 mutations co-occurred more commonly with DNMT3A, NPM1, and FLT3 internal tandem duplication mutations. Compared with PTPN11 wild-type (WT) patients, PTPN11 mutation-positive AML patients presented with higher white blood cell (WBC) and platelet (PLT) counts. In 74 PTPN11 positive AML patients, PTPN11 mutations had an adverse effect on overall survival (OS) (62.5%) and a negative prognostic effect on event-free survival (EFS) (50%). Allo-hematopoietic stem cell transplantation (HSCT) abrogated the negative effect of mutations in PTPN11; the OS and EFS of AML patients with PTPN11 mutations who received transplantation were longer than those of AML patients with PTPN11 mutations who did not undergo allo-HSCT (P = 0.001, EFS; P < 0.001, OS). Discussion: Newly diagnosed PTPN11 mutation-positive AML patients with high WBC and PLT counts or presenting no remission after first induction chemotherapy suffer from high mortality rates.

Conclusion:

Given the lack of targeted therapies for PTPN11 mutations, timely HSCT is necessary for patients.

KEYWORDS:

• PTPN11 mutations
• acute myeloid leukemia
• secondary mutation
• prognosis factor

Introduction

Acute myeloid leukemia (AML) is a clinically and biologically heterogeneous disease with variable responses and survival outcomes [1,2]. Over the last decade, better characterization and understanding of the AML genomic landscape have led to significant progress in prognostication and treatment [3,4].

RAS is a proto-oncogene belonging to the family of small GTPases and is vital for cellular signal transduction [5]. RAS activation leads to increased signaling through the RAS/RAF/MEK and RAS/PI3K pathways [6]. RAS has three isoforms: NRAS, KRAS, and HRAS, and mutations in these can activate RAS mutations. In addition to directly activating RAS mutations, other gene mutations (e.g. FLT3, KIT, and CBL) upstream of RAS in the signaling cascade or gene mutations (e.g. PTPN11 and NF1) involved in RAS signaling regulation can also activate RAS signaling pathways [7].

PTPN11, located on chromosome 12q24, encodes a protein with Src-homology 2 (SH2) and a tyrosine phosphatase domain [8,9]. The SH2 domain is required for normal development and is an essential component of signaling pathways initiated by growth factors, cytokines, and the extracellular matrix [10–12]. Moreover, the SH2 domain is a conformational switch; it either binds and inhibits phosphatases or binds phosphoproteins to activate the enzyme [13,14]. Mutations affecting amino acid residues on the interacting surfaces of these domains lead to a gain of function and alter protein activity. Germline and somatic mutations of PTPN11 are known to occur in children with Noonan syndrome [15,16] and juvenile myelomonocytic leukemia [17–19]. However, little is known about the clinical impact of these mutations in adult acute leukemia [19–21]. Until recently, reports showed that 8% of patients with AML had PTPN11 mutations [8]. PTPN11 mutations co-occur more often with NPM1 mutations and FLT3-ITD mutations, less often with mutations in IDH2 and in those with a complex karyotype, and are related to lower complete response rates and shorter overall survival [2,22].

To evaluate the impact of PTPN11 mutations on clinical outcomes, patients with AML were screened using targeted next-generation sequencing (NGS), acknowledging the association between NPM1 mutations and poor prognosis in patients with AML.

Materials and methods

Patients

This study included 74 patients with AML diagnosed between June 2016 and June 2020 at the Department of Hematology in the First Affiliated Hospital of Soochow University with targeted NGS panels that included the PTPN11 gene. All patients harboring genetic alterations were diagnosed according to the World Health Organization (WHO) criteria for the diagnosis of AML. The study was approved by the Ethics Committee of the First Affiliated Hospital of Soochow University (No. 164 of 2020 LSP [application]) and conducted in accordance with the Declaration of Helsinki.

Targeted NGS

Genomic DNA was extracted from bone marrow (BM) aspirates from 74 patients at diagnosis. Each DNA library was then sequenced using an Illumina X Ten for paired-end reads of 150 bp using the targeted gene panel design, which was intended to cover the myeloid tumors’ common gene regions involved in signal transduction, splicing, transcription, and epigenetic modification. The NGS panels include genes that are frequently affected by hematologic malignancies (Supplementary Material 1). A minimum sequencing coverage of 500 × (bidirectional true paired-end sequencing) is required. Analytical sensitivity was established at 5% mutant reads on a background of wild-type (WT) reads. The variant allele frequency (VAF) was determined as the ratio of mutant to total (WT + mutant) reads. Genetic analysis of non-hematopoietic tissues of patients or peripheral blood of the patients’ parents was of critical importance, additional to the screening of leukemic cells, to determine whether the mutation was somatic or germline, considering the milder clinical course described in germline mutations. In instances of potential leukocyte contamination of hair follicles or buccal swab samples, fingernail or parent samples were collected [23].

Statistical analysis

Patient outcome data updated on November 31, 2020 were used. The overall survival (OS) time was considered from diagnosis to death. Event-free survival (EFS) time was calculated from diagnosis to first failure, including death, relapse, or treatment abandonment due to disease progression. EFS and OS curves were estimated using the Kaplan-Meier method and compared using the log-rank test. Categorical variables were compared using chi-square or Fisher exact tests. Continuous variables were compared using the Wilcoxon rank-sum test. All P values were 2-sided, with values < 0.05 indicating statistical significance. SPSS Mac23.0.0 (IBM Corp., Armonk, NY, USA) software was used for statistical analysis.

Results

Baseline characteristics

Among the 74 patients enrolled with de novo AML, 35 were male and 39 were female. The median age was 43 years (range, 6–68). The median WBC at diagnosis was 24.05 (1.22–248)    10⁹/L, the median platelet count was 60 (5–713)    10⁹/L, and the median hemoglobin concentration was 81 (35–149) g/L. Moreover, the median percentage of BM blasts was 62.5% (range, 13–95%). Among the 1101 PTPN11 mutation-negative AML patients (the no-PTPN11 mutation group), 590 were male and 511 were female. The median age was 43 years (range, 3–79). The median WBC at diagnosis was 13.33 (0.29–584.1)    10⁹/L, median PLT count was 42 (1–713)    10⁹/L, and median hemoglobin level was 85 g/L (range, 16–165 g/L). The median percentage of BM blasts was 56% (range, 3–99.5%). For recurrent genetic abnormalities, PTPN11 mutations were detected in patients with fusion genes, such as FUS::ERG, DEK::NUP214, BCR::ABL, and MLL rearrangement. All mutations were evaluated using multiple databases (Supplementary Material 2).

Landscape of PTPN11 mutations

Up to 99 genotypes of PTPN11 mutations were detected in the 74 patients. Most of these mutations involve either N-SH2 or protein tyrosine phosphatase (PTP) domains, similar to reports from pediatric cohorts [24]. The incidences of the mutated loci were E76 (19/99, 19.2%), D61 (16/99, 16.2%), A72 (12/99, 12.1%), S502 (8/99, 8.1%), and G503 (7/99, 7.1%) (Figure 1). In these mutations, D61, A72, and E76 are in the N-SH2 domain, and S502 and G503 are in the PTP domain. Most mutations in these patients were missense mutations.

Figure 1. Distribution of mutation sites of PTPN11 mutations. The number in the circle represents the number of mutations, with the most commonly mutated loci being E76 (19/99, 19.2%), D61 (16/99, 16.2%), A72 (12/99, 12.1%), G503 (7/99, 7.1%), and S502 (8/99, 8.1%).

Among the 74 patients with AML, PTPN11 mutations commonly co-occurred with NPM1 (24/74, 32.4%, P < 0.001), DNMT3A (22/74, 29.7%, P < 0.001), and NRAS mutations (20/74, 27.0%, P < 0.001), but were rarely observed with CEBPA (4/74, 5.4%, P = 0.06), KIT (2/74, 2.7%, P = 0.056), or EZH2 mutations (2/74, 2.7%, P = 0.073) compared to those in PTPN11 mutation-negative patients (Table 1 and Figure 2). However, there was no significant difference in FLT3-ITD mutations (P = 0.136).

Figure 2. Catastrophe distribution waterfall of acute myeloid leukemia (AML) with PTPN11 mutations. DNMT3A mutation rate in PTPN11 mutation-positive AML patients was 29.7% (22/74); the FLT3-ITD mutation rate was 25.6% (19/74), and the NPM1 mutation rate was 32.4% (24/74).

Table 1. Baseline characteristics of PTPN11 mutation-positive acute myeloid leukemia patients.

Characteristics	PTPN11(N = 74)	NO-PTPN11(N = 1101)	P-value
Age (year. range)	43(6–68)	43(3–79)	0.959
Sex (male/female)	35/39	590/511	0.294
WBC (*10^9/L. range)	24.05(1.22–248)	13.33(0.29–584.1)	0.003**
Hemoglobin (g/L. range)	81(35–149)	85(16–165)	0.732
PLT (*10^9/L. range)	60(5–713)	42(1–713)	0.011*
Bone marrow blasts (%)	62.5(13–95)	56(3–99.5)	0.278
Cytogenetics—no.
Normal	44(59.5%)	540(49.0%)	0.083
others	30(40.5%)	456(41.4%)	0.882
No phase	0(0%)	105(9.5%)	0.001**
Fusion gene	17(23.0%)	312(28.3%)	0.320
Mutation			56154
CEBPA	4(5.4%)	142(12.9%)	0.06
FLT3-ITD	19(25.7%)	206(18.7%)	0.136
NPM1	24(32.4%)	117(10.6%)	<0.001**
DNMT3A	22(29.7%)	147(13.4%)	<0.001**
NRAS	20(27.0%)	128(11.6%)	<0.001**
KIT	2(2.7%)	103(9.4%)	0.056
EZH2	2(2.7%)	53(4.8%)	0.073
TET2	6(8.1%)	113(10.3%)	0.558
ASXL1	5(6.8%)	61(5.5%)	0.601

PTPN11: PTPN11 mutation-positive AML patients; NO-PTPN11: PTPN11 mutation-negative AML patients; no. number; WBC: white blood cell counts; PLT: platelet counts.

*p < 0.05.

**p < 0.01.

Impact of PTPN11 mutations on response and survival

In the newly diagnosed cohort, the median age of patients with AML and PTPN11 mutations was 43 years and the median BM blast cell rate was 62.5% (Table 1). Compared with PTPN11 mutation-negative AML patients who were also diagnosed between June 2016 and June 2020 at our clinical center, patients with newly diagnosed AML and PTPN11 mutations presented with high WBC and PLT counts and higher rates of NPM1, NRAS, and DNMT3A mutations (Table 1). There were no differences in sex, hemoglobin, cytogenetic type, fusion gene number, or FLT3-ITD mutation between the PTPN11 mutation group and the no-PTPN11 mutation group.

Next, we investigated the impact of clinical variables on the survival of patients with PTPN11 mutations. AML patients with PTPN11 mutations had a median EFS of 0.57 years and median OS of 0.73 years. The Kaplan-Meier method analysis for OS in AML patients with PTPN11 mutations revealed significant differences in OS of patients with ASXL1 mutation, with one-course complete remission, and in patients who underwent transplantation. Although NPM1 mutation was observed in nearly 30% of PTPN11 mutations AML cases, the clinical outcome of these patients was not affected (P = 0.604). The Kaplan-Meier analysis for EFS in AML patients with PTPN11 mutations showed a significant difference in EFS of patients who had one-course complete remission and in those who underwent transplantation. DNMT3A mutation was seen in over 30% of PTPN11 mutant AML cases; however, the EFS was not affected (P = 0.065).

Multivariate analysis of survival in AML patients

To assess the relative contribution of PTPN11 mutations in evaluating prognosis, we performed a univariate analysis in which baseline characteristics were included. We found that high WBC count, high BM blast rate, first induction chemotherapy no remission, and coexisting with ETV6, ASXL1, or TET2 mutations increased the risk of death in newly diagnosed AML patients with PTPN11 mutations (Table 2). Multivariate analysis identified high WBC count, and first induction chemotherapy no remission as factors predicting an increased risk of death (Table 3). Receiving allo-HSCT was associated with a lower risk of death.

Table 2. Univariate analysis of the OS analysis in PTPN11 mutations positive AML patients.

Characteristics	N/total(N = 74)	HR (95%CI)	P-value
Age (year. range) <43 >43	37 37	1.497(0.699–3.208)	0.300
Sex (male/female) Female Male	39 35	0.757(0.354–1.620)	0.474
WBC (*10^9/L. range) <24.05 >24.05	36 36	2.594(1.141–5.897)	0.023*
Hemoglobin (g/L. range) <81 >81	36 37	1.746(0.770–3.958)	0.182
PLT (*10^9/L. range) <60 >60	36 37	1.017(0.464–2.231)	0.966
Bone marrow blasts (%) <62.5 >62.5	33 33	2.074(0.905–4.754)	0.085
Cytogenetics—no. Normal others	44 30	1.073(0.502–2.293)	0.856
Fusion gene positive negative	17 57	2.016(0.931–4.366)	0.075
Induction chemotherapy CR NO CR	44 30	0.321(0.150–0.689)	0.004**
Bone marrow transplant YES NO	41 33	0.185(0.082–0.418)	<0.001**
Relapse YES NO	17 57	1.048(0.457–2.404)	0.912
ASXL1 YES NO	5 69	3.393(1.006–11.435)	0.049*
ETV6 YES NO	2 72	58.369(8.053–423.081)	<0.001**
TET2 YES NO	6 68	4.460(1.222–16.276)	0.024*

OS: overall survival; N: number; HR: Hazard Ratio; WBC: white blood cell counts; PLT: platelet counts; no.: number; CR: complete remission; NO CR: no complete remission.

*P < 0.05.

**P < 0.01.

Table 3. Multivariate analysis of the OS analysis in PTPN11 mutations-positive AML patients.

Characteristics	N/total(N = 74)	HR (95%CI)	P-value
WBC (*10^9/L. range) <24.05 >24.05	36 36	4.953(1.517–16.173)	0.008**
Bone marrow blasts (%) <62.5 >62.5	33 33	2.041(0.782–5.326)	0.145
Fusion gene positive negative	17 57	2.835(0.970–8.289)	0.057
Induction chemotherapy CR NO CR	44 30	0.185(0.060–0.573)	0.003**
allo-HSCT YES NO	41 33	0.385(0.148–1.001)	0.050
ASXL1 YES NO	5 69	0.224(0.019–2.688)	0.238
ETV6 YES NO	2 72	15.697(0.857–287.559)	0.063
TET2 YES NO	6 68	9.022(1.683–48.362)	0.010*

OS: overall survival; N: number; HR: Hazard Ratio; WBC: white blood cell counts; PLT: platelet counts; CR: complete remission; NO CR: no complete remission.

*P < 0.05.

**P < 0.01.

Impact of PTPN11 mutations on transplant outcomes

Among the 74 newly diagnosed patients in the study cohort, 40 (54%) underwent allo-HSCT. In a landmark analysis, we compared OS by transplant status and PTPN11 mutational status. Results showed that AML patients with PTPN11 mutations who underwent allo-HSCT had a median EFS of 1.3 years and median OS of 1.36 years. A landmark survival analysis to investigate whether allo-HSCT abrogates the adverse impact of mutations in PTPN11 was performed, wherein we compared patients who underwent allo-HSCT (N = 41) with those who did not (N = 33) among AML patients with PTPN11 mutations. The analysis revealed no significant differences in survival outcome. The OS and EFS of AML patients with PTPN11 mutations who underwent transplantations were longer than those in patients with PTPN11 mutations who did not undergo allo-HSCT (P = 0.001 and P < 0.001 for EFS and OS, respectively, Figure 3).

Figure 3. Kaplan-Meier overall survival and event free survival curves of acute myeloid leukemia (AML) with PTPN11 mutations showed that the patients who underwent transplantation. A: overall survival; B: event free survival.

Discussion

This study aimed to investigate the clinical characteristics and prognostic impact of PTPN11 mutations in AML patients. Our analysis of patients with AML revealed a PTPN11 mutation frequency of 4%, similar to that reported in a Brazilian cohort [25,26]. In addition, PTPN11 mutations occurred more often in males (77.8%). However, we did not find a relationship between PTPN11 mutations and patient sex. Maria et al. reported that mutations in PTPN11 negatively impacted the survival of pediatric patients with AML [24]. Our results led us to conclude that PTPN11 mutations result in poor outcomes in both pediatric and adult AML patients [27–29].

Our study investigated a cohort of PTPN11 mutation-positive acute leukemia cases. Results revealed that PTPN11 mutation was a predictor of poor prognosis in PTPN11 mutation-positive AML patients. Newly diagnosed AML patients with PTPN11 mutations who presented with high WBC counts, and no remission after the first induction chemotherapy, had particularly high mortality rates. Considering the number of TET2 mutation positive patients was very small and the credibility of the results was greatly reduced, we were cautious with the conclusion about TET2 mutations.

Remarkably, most PTPN11 mutations found in our study were missense. The most common mutations were D61, A72, and E76 in the N-SH2 domain, and S502 and G503 in the PTP domain. PTPN11 mutations commonly co-occurred with DNMT3A, FLT3-ITD, and NPM1 mutations in patients with AML, implying that these mutations may cooperate in generating this specific leukemic phenotype. Conversely, in PTPN11 mutation-positive AML patients with fusion genes (FUS::ERG, DEK::NUP214, BCR::ABL, and MLL rearrangement), less NPM1 mutations were detected. Previous studies reported that ∼85% of patients with NPM1 mutated AML have a normal karyotype, while the remaining (∼15%) patients present with mostly minor chromosomal abnormalities that are likely secondary events [30]. However, almost all fusion genes are formed by chromosomal ectopic, and the existence of chromosomal abnormalities suggests that this might be the cause of the lower rate of NPM1 mutations in PTPN11 mutation-positive AML patients with fusion genes.

Swoboda et al. and Fobare et al. suggested that HSCT significantly improves outcomes in AML patients with PTPN11 mutations and is highly recommended after induction chemotherapy [2,31]. Our results showed that in AML patients with PTPN11 mutations, the OS and EFS of patients who underwent transplantation were longer than that in patients who did not (P = 0.001 and P < 0.001 for EFS and OS, respectively). Given the limited number of cases in this study, this conclusion should be further verified through a multi-center study with a larger number of patients.

We analyzed the clinical and molecular characteristics along with the clinical outcomes of a large cohort of PTPN11 mutation-positive AML patients. Although the number of patients included in our cohort was large, the samples were considered negligible due to the low incidence of PTPN11 mutations, which could affect the broader application of our results. A larger series is necessary to validate these findings. In conclusion, the overall outcome and prognosis of PTPN11 mutation-positive patients appear poor; therefore, we recommend transplantation as soon as possible. New therapies are needed to target this high-risk subtype of AML.

Acknowledgments and funding

This study was supported by grant from the National Key R&D Program of China (2019YFA0111000), the National Natural Science Foundation of China (82100170, 82000158), the Natural Science Foundation of the Jiangsu Higher Education Institution of China (18KJB320019), the Natural Science Foundation of Jiangsu Province (BK20210087), priority academic program development of Jiangsu Higher Education Institution, the Innovation Capability Development Project of Jiangsu Province (BM215004), the Open Project of Jiangsu Biobank of Clinical Resources (SBK202003001, SBK202003003), Jiangsu Provincial Key Medical Center (YXZXA2016002) and National Science and Technology Major Project (2017ZX09304021). All the samples were from Jiangsu Biobank of Clinical Resources.

Data availability statement

The data that supports the findings of this study are available in the supplementary material of this article.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Additional information

Funding

This work was supported by National Natural Science Foundation of China: [Grant Number 82000158]; National Natural Science Foundation of China: [Grant Number 82100170]; Natural Science Foundation of Jiangsu Province: [Grant Number BK20210087].