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| ORIGINAL ARTICLE |
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| Year : 2022 | Volume
: 2
| Issue : 2 | Page : 102-107 |
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Evaluation of morphological, clinical, and prognostic significance of nucleophosmin gene mutation in de novo acute myeloid leukemia: A retrospective study
Sudarshan Venkat Pandit1, Nilesh Wasekar1, Girish Badarkhe1, Rajat Bajaj1, Yasam Venkata Ramesh2, Raj Nagarkar3
1 Department of Hematology, HCG Manavata Cancer Centre, Nashik, Maharashtra, India
2 Department of Academics, HCG Manavata Cancer Centre, Nashik, Maharashtra, India
3 Department of Surgical Oncology, HCG Manavata Cancer Centre, Nashik, Maharashtra, India
| Date of Submission | 31-Jan-2022 |
| Date of Decision | 10-Jun-2022 |
| Date of Acceptance | 04-Oct-2022 |
| Date of Web Publication | 06-Feb-2023 |
Correspondence Address:
Dr. Sudarshan Venkat Pandit
Department of Hematology, HCG Manavata Cancer Centre, Nashik - 422 011, Maharashtra
India
 Source of Support: None, Conflict of Interest: None
DOI: 10.4103/jpo.jpo_2_22
Introduction: Nucleophosmin 1 (NPM1) is a molecular chaperone protein. Mutation of NPM1 gene is identified to be one of the most predominant molecular abnormalities in acute myeloid leukemia (AML). Among the genetic mutations, NPM1 along with FMS-like tyrosine kinase 3 (FLT3) mutations lead to poor prognosis and unfavorable outcomes in patients with AML.
Methods: This was a retrospective study conducted for 2 years at a leading cancer care center in India. A total of 10 patients were identified to be NPM1 positive in 50 identified AML patients.
Results: In 10 NPM1-positive patients, eight patients were female. All were adults with a median age of 39.3 years. The majority of the patients were presented with Auer rod (7/10) and intranuclear cup-like inclusions (7/10) with increased number of blasts in peripheral blood smear (77.88%). Lymphadenopathy was absent in all patients. Morphological analysis has shown blast cells with a cup shape and prominent nucleoli with indentation. Whereas, immunophenotyping analysis has shown lower positivity of cluster of differentiation 34 (12%) and lack of human leukocyte antigen-DR expression (34%).
Conclusion: The presence of NPM1 mutations alone without FLT3 mutations in patients with AML was observed to have a better prognosis of the disease. Early identification of such mutations by morphological and immunophenotyping analysis can play a vital role in treatment initiation at the earliest for better prognosis and clinical outcomes without much delay.
Keywords: Acute myeloid leukemia, FMS-like tyrosine kinase 3, immunophenotyping, nucleophosmin 1, nucleophosmin, prognosis
How to cite this article:
Pandit SV, Wasekar N, Badarkhe G, Bajaj R, Ramesh YV, Nagarkar R. Evaluation of morphological, clinical, and prognostic significance of nucleophosmin gene mutation in de novo acute myeloid leukemia: A retrospective study. J Precis Oncol 2022;2:102-7 |
How to cite this URL:
Pandit SV, Wasekar N, Badarkhe G, Bajaj R, Ramesh YV, Nagarkar R. Evaluation of morphological, clinical, and prognostic significance of nucleophosmin gene mutation in de novo acute myeloid leukemia: A retrospective study. J Precis Oncol [serial online] 2022 [cited 2023 Mar 28];2:102-7. Available from: https://www.jprecisiononcology.com//text.asp?2022/2/2/102/369214 |
| Introduction | |  |
Acute myeloid leukemia (AML) is the most common form of acute leukemia (AL) in adults.[1] Malignant transformation of immature hematopoietic cells characterized by impaired differentiation and autonomous proliferation will develop as AML. Molecular and cytogenetic analyses are currently used to risk-stratify AML.[2] Several molecular and gene level abnormalities were identified with AML, which includes a recurrent fusion of genes such as t (8;21), t (15;17), and inv (16), which are ultimately hindering the differentiation and self-renewal process.[2],[3] Change in genes associated with proliferation and survival potential of hematopoietic progenitors was also observed to be highly mutated, which include nucleophosmin 1 (NPM1), oncogenic transcription factor (CEBP/alpha), and receptor tyrosine kinases (FMS-like tyrosine kinase [FLT3], KIT, and RAS).[4] Understanding such genetic abnormalities can help in knowing the AML mechanism, risk stratification, response assessment, and the development of individualized therapies.[5]
In a study conducted by Falini et al.[1] and Verhaak et al.[2], it was observed that approximately 35% of de novo AML cases were due to the NPM1 exon 12 mutations.[1],[2] It is also recognized as one of the most frequent mutations in AML patients with a normal karyotype.[1],[2] NPM1 plays a vital role as a versatile partner in many chromosomal translocations and it is the most explored gene of the NPM family. NPM1 is a ubiquitously expressed phosphoprotein, which is referred to as NO38 or numatrin, or nucleolar protein B23, respectively. NPM1 is primarily located in the nucleolus, functioning as a molecular chaperone of proteins by continuously shuttling between the cytoplasm and nucleus, facilitating the transport of ribosomal proteins through the nuclear membrane. Any disruption of NPM1 either by mutation or by chromosomal translocation results in cytoplasmic dislocation. The NPM1 also has several other functions such as regulation of centrosome function, maintenance of genomic stability, binding of p53, preventing aggregation of proteins in the nucleolus, and controlling the alternate reading frame protein adenosine diphosphate-ribosylation factor-P53 tumor-suppressor pathway.
Many studies have indicated the NPM1 mutation and its role in the prognosis and final outcome for AML.[1],[6],[7],[8] More recently, the prognostic effect of NPM1 mutations and FLT3 internal tandem duplication (ITD) in AML have become well established and multiple studies have confirmed their coexistence in approximately 40% of AML patients. In most of the patients, FLT3-ITD is associated with higher blast counts and white blood cell counts at diagnosis indicating poor prognosis.[9],[10],[11],[12] Whereas, patients with NPM1 mutation alone without FLT3-ITD were observed to have a better prognosis, disease-free survival (DFS), and overall survival (OS) with a lower relapse rate compared to those patients who had NPM1 mutation along with FLT3-ITD.[6],[9],[10],[11],[12],[13]
There have been very few studies in the Indian population examining the prevalence and clinical characteristics of NPM1 mutations in AML. The frequency of NPM1 mutation was also observed to be low in the Indian population compared to the Western population.[14]
The aim of this study was to investigate and evaluate the morphological features, immunophenotyping, clinical characteristics, prognosis, and treatment outcomes of NPM1 mutations in de novo AML patients of Indian origin.
| Methods | | |
The retrospective study was conducted between January 2018 and January 2021 and 50 cases of AML were identified. Written informed consent was obtained from all patients prior to the study commencement.
At the time of diagnosis, clinical data were collected from all the patients. Aspirations/biopsies of bone marrow were performed on the same day, and peripheral blood smears were obtained to confirm the diagnosis. All the evaluations were done as per the French–American–British criteria. From the collected samples, immunophenotyping studies by flow cytometry and mutation analysis were performed.
Immunophenotyping studies were carried out using various monoclonal antibodies (from Beckman Coulter Life Sciences) in the panel to gate the blast cell population using the Beckman Coulter DxFlex-6 color instrument using Kaluza Analysis Software (Beckman Coulter, USA).[4]
Upon confirmation of AML, chemotherapy was initiated for all of our patients according to guidelines following their Eastern Cooperative Oncology Group (ECOG) status and family's consent.[4],[15] Further, mutation analysis was performed using Next-generation Sequencing (NGS) on the Illumina MiSeqDx, DNA, and polymerase chain reaction sequencing for NPM1 exon 12 mutation and FLT3, respectively.[16],[17]
Treatment protocol
Chemotherapy treatment in patients with AML was typically divided into two phases: remission induction and consolidation.[4],[15]
The first remission induction phase of the treatment is quickly aimed at decreasing leukemia cell load in the patients. In this phase, treatment options were highly dependent on the patient's age, ECOG status, and willingness of the family. The standard treatment protocol in this phase is “7 + 3” (cytarabine and anthracycline) induction chemotherapy or all-trans-retinoic acid therapy. The second phase (consolidation phase) consisted of three courses of high-dose cytosine arabinoside followed by granulocyte colony stimulating factor (GCSF) according to the condition of the patient. After 28 days of treatment initiation, bone marrow aspiration was done to evaluate the response to treatment.
| Results | | |
The clinical and hematological characteristics of the patient's cohort are summarized in [Table 1]. In a sample of 50 adult patients, 10 were positive for NPM1, and 40 were negative for the protein. There were eight female patients and two male patients. They had a median age of 39.3 years. Hepatosplenomegaly was observed in 2 of 10 patients. Typical morphological characteristics were observed in blast cells such as cup shape, granular, blast with prominent nucleoli, and indentation [Figure 1]. | Figure 1: Peripheral blood smear demonstrating cup-shaped blast (a), blast with prominent nucleoli (b), blast with indentation (c), granular blast (d and f), and cup-shaped blast (e)
Click here to view |
Whereas, immunophenotypic characteristics of all the NPM1-positive and NPM1-negative patients are presented in [Table 2]. Where an association of NPM1 mutation with lower positivity of cluster of differentiation 34 (CD34) (12%) and lack of human leukocyte antigen-DR (HLA-DR) expression (34) was identified [Figure 2]. Among 10 NPM1-positive patients, one patient underwent mutation analysis, where c.863_864insTCTG (p. W288fs*12) NPM1 exon 12 mutation was identified. | Table 2: Immunophenotypic characteristics of acute myeloid leukemia patients with nucleophosmin 1 mutations
Click here to view |
 | Figure 2: Flow cytometry showing blast population which is CD 34 negative (a), CD 117 positive (b), Anti-HLA-DR negative and CD 36 positive (c), CD 33 positive (d), CD 36 and CD 64 positive suggestive of monocytoid differentiation (e), and Cy myeloperoxidase positive (f). CD: Cluster of differentiation, HLA-DR: Human leukocyte antigen-DR
Click here to view |
Due to economic constraints, NGS was performed in only one NPM1-positive patient. Where 40 key DNA target genes and 29 driver genes in a broad fusion panel were covered to determine all the major myeloid disorders and several acute lymphoid disorders. Finally, it was reported as a known case of AML with IDH1 mutation (locus-chr2:209113112; mutation-c.394C>T (p.Arg132Cys).
Seven (70%) of the 10 NPM1-positive patients had undergone standard 7 + 3 induction chemotherapy (cytarabine, 100 mg/m2 per day continuously for seven days, as well as short infusions of an anthracycline on each of the first three days), while 3 (30%) had received azacitidine-based chemotherapy. After induction chemotherapy, two patients with NPM1 positive and NPM1/FLT3 positive were succumbed to death due to sepsis.
After confirming the AML-minimal residual disease (AML-MRD) negativity, the remaining eight patients were kept on consolidation chemotherapy using high-dose cytosine arabinoside (every 12 h on days 1, 3, and 5 at 3 g/m2 in a total of 18 g/m2) followed by G-CSF (5 μg/kg daily from day 8 till counts were recovered.[18],[19] Overall, six patients had completed their consolidation chemotherapy. Postconsolidation chemotherapy, AML-MRD, and NPM1 mutation status were evaluated and reported to be negative. The remaining two FLT3-positive patients were initiated individually on sorafenib (multikinase inhibitor) and midostaurin (tyrosine kinase inhibitor).[20] Among those two patients, one patient is still in the consolidation phase and one patient is receiving azacitidine-based maintenance chemotherapy. As of date, both patients are continuing their chemotherapy while waiting for their hematopoietic stem-cell transplant. All patients were kept on regular observation and follow-up.
| Discussion | | |
In the WHO 2016 Classification, NPM1 positive AML has been categorized as a good-risk disease. The present retrospective study identified 10 cases with NPM1 positive AML. In order to assess the prognostication features of patients with AML, morphological characteristics, flow cytometric patterns, and prognostication characteristics have been analyzed and evaluated. Many studies have been conducted over the years to identify the role of NPM1 and FLT3 in AML prognosis. The prognostic implications of such NPM1 mutations were generally ambiguous and susceptible to bias.
The rate of NPM1 mutation in our study cohort was reported to be 20%, similar to the frequency reported in German (27.4%), Japan (24.9%), and Liu et al.[5] (6% to 56%) in the adult AML population. Such large variation in NPM1 mutation between the studies can be attributed to the number of cases studied, the group of population in which it was conducted, and the geographical distribution of the population.[5]
Findings from previous studies have also suggested the prognostic significance of NPM1 mutation and its favorable effect on remission rate, DFS, OS, and outcome of patients with AML.[2],[5],[6],[10] Whereas, FLT3 mutation is another important abnormality in AML patients with marked poor outcomes with reduced survival, increased risk of relapse from complete remission (CR), and a high percentage of bone marrow blast cells.[5],[21]
Hence, the combination of NPM1 and FLT3 mutation and its prognostication on a patient's survival outcomes have attained much importance.
Age is a strong prognostic factor in AML patients. Multiple studies have confirmed the striking relationship between age, incidence of NPM1 mutation, and prognosis. The frequency of NPM1 mutations was observed to be increasing with age.[1],[22] Age-related incidence was evident in our study cohort, where all the identified AML patients were adults in accordance with the previous studies.[4],[14] NPM1 mutations were also noticed to be high in a female population over males, which aligns with the results from Gale et al.[9]
A peculiar morphological feature was observed in NPM1-positive AML patients. Where a majority of the patients had myelomonocytic pictures along with blasts having prominent nucleoli, indentation with azurophilic granules, and cup-shaped blasts.
Immunophenotyping analysis results of our patients were also in accordance with earlier studies revealing the association between NPM1 mutation and lack of HLA-DR and CD34 expression, respectively.[23],[24] The lack of CD34 expression in NPM1-positive patients was found to be an indicator for a good response to induction chemotherapy, and it will help the patients positively.
The current treatment paradigm initially employs remission-inducing chemotherapy in AML patients with “7 + 3” (cytarabine and anthracycline) regimen, followed by consolidation therapy.[25],[26] Over the past four decades, this treatment regimen is achieving CR in 60%–80% of patients under <60 years of age.[11],[27],[28],[29] Similar results were noted in our study cohort with 60%–70% of patients responding to treatment with a CR, while patients with FLT3 mutations were categorized as high risk with a poor prognosis. These cases were addressed by adding new therapeutic agents such as sorafenib[30],[31] and midostaurin[31], as well as standard-of-care chemotherapy “7 + 3” for improved outcomes. In phase 3 clinical trial conducted by Stone et al.[32], the addition of midostaurin has significantly prolonged overall and event-free survival among FLT3-positive AML patients, and the same was observed in our patients.[32] In another nonrandomized trial conducted by Röllig et al.[30] adding sorafenib to standard chemotherapy had shown better antileukemic efficacy but with increased toxicity.[30] For the patient's overall benefit, both additional drugs were used in our patients with FLT3 mutations along with their standard of chemotherapy for better outcomes. A possible advantage of these additional drugs is their ability to block the autophosphorylation of the FLT3 kinase, leading to the inhibition of cell proliferation and induction of apoptosis.[33]
To summarize, NPM1 mutations were found to be high in females, aged >40 years, with distinct morphological and flow cytometry characteristics. As a clinician, identification of such distinct features can help in the initiation of therapy at the earliest without much delay. However, in future, research should focus on identifying the mechanistic link and investigating the interaction between these frequent NPM1 and FLT3 mutations in AML patients. This can help both patients and clinicians to gain better insight into the pathogenesis of AML, giving better individual-tailored therapeutic treatment, and ultimately helping in improving the overall prognosis and survival of the patients.
The main limitation of this study is its retrospective nature and a low number of patients due to the rarity of the condition. Limiting factors were investigated to identify the association between the morphological, clinical, and prognostic significance in patients with NPM1-positive AML. Extensive mechanistic link identification between the NPM1 and FLT3 mutations in a large population is warranted.
| Conclusion | | |
NPM1 mutations alone in patients with AML were observed to have a better prognosis over patients with NPM1 and FLT3 mutations. Gender, age, morphological, and flow cytometry results can play a vital role in providing initial data to clinicians for treatment initiation at the earliest for better prognosis and clinical outcomes.
Acknowledgment
The authors would like to thank Dr. Yasam Venkata Ramesh from HCG Manavata Cancer Centre, Centre for difficult cancers, Nashik, India, for his medical writing assistance.
Financial support and sponsorship
Nil.
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2]
[Table 1], [Table 2]
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