PhD Student of Hematology, Blood Transfusion Research Center, High Institute for Research and Education in Transfusion Medicine, Tehran, Iran

Background: Early detection of hematologic relapse and prompt management of acute myeloid leukaemia (AML) patients are made possible by minimal residual disease (MRD) diagnostics. AML patients who do not have clonal chromosomal abnormalities are classified as having cytogenetically normal acute myeloid leukaemia in about 50% of cases (CNAML). Exon 12 of the NPM1 gene is mutated in about 60% of adult CN-AML cases. This mutation is unique to malignant clones and may be an effective MRD marker. In this retrospective investigation, we followed up on AML patients who had the NPM1 type A mutation at the time of diagnosis and set up a quantitative assay to measure it.

Materials and Methods: Using PCR cloning, we created plasmids with cDNA fragments for the NPM1 and ABL genes. The standard curves were built using plasmids. Eleven patients underwent analysis utilising a tried and true procedure. Serial PB and/or BM samples (n=71) were collected at intervals of 1-3 months (mean 1.5 months), and the median follow-up time following treatment was 11 months (5-28.5 months)

Conclusion: Despite the study's sample size and follow-up period constraints, it demonstrated the accuracy of the setup for mutation detection and demonstrated the use of this marker for monitoring disease progression at various stages. NPM1 is an appropriate marker for monitoring NPMc+ AML patients due to its high frequency, stability, specificity to aberrant clones, and high sensitivity. Keywords: NPM1 mutation, MRD, acute myeloid leukaemia

Acute myeloid leukemia describes by abnormal proliferation of hematopoietic precursors and disturbing the normal hematopoiesis and it is the clinically, cytogenetically and molecularly heterogeneous disorder. AML patients are categorized in three risk groups favorable,intermediate, or unfavorable) based upon cytogenetic risk factors. The remarkably heterogeneous nature of cytogenetically normal acute myeloid leukemia (CNAML) has become more apparent by discovery of multiple molecular lesions in this group of patients that results in difference in response to chemotherapy and clinical outcome.

About 30% of AMLs and 50%–60% of CN-AML had NPM1 mutations, the most prevalent genetic aberration found in adult de novo AML.The NPM1 gene may be found in band q35 on the long arm of chromosome NPM1 is a nucleocytoplasmic trafficking protein that is localised in the nucleolus. Transport of ribosomal components into the cytoplasm, regulation of centrosome duplication, interaction with p53, and modulation of the ARF-Hdm2/Mdm2-P53 oncogenic suppressor pathway are among NPM1's key roles.

The most prevalent NPM1 mutation, type A, results from a tetra-nucleotide insertion (TCTG) at position 956–959 of the NPM1 gene and is observed in around 80% of patients of NPMc+ AML. The NPM1 mutations are found in exon .In order to divide this heterogeneous group of AML patients into prognostically distinct subgroups, molecular markers are helpful.This mutation is unique to malignant clones and may be a reliable indicator of MRD.The initial objective of treatment for AML patients is to achieve hematologic full remission.Most AML patients relapse three to five years following diagnosis. Minimal residual disease (MRD) tests allow for prompt treatment of patients with acute myeloid leukaemia (AML) and early detection of hematologic relapse.RT-PCR quantitative technique is the approach with the highest sensitivity (1×10-5) for identifying MRD.AML patients that do not have clonal chromosomal abnormalities are classified as having cytogenetically normal acute myeloid leukaemia (CNAML) in about 50% of cases, whereas 30- 40% of patients with CN-AML had FLT3/ITD mutations. FLT3/ITD mutations are a contentious MRD marker since they are thought to be a somewhat unstable signal that may be lost upon relapse.

The presence of disease in AML patients is related with high levels of WT1 expression in BM and PB, and a rise in WT1 levels prior to haematological recurrence may indicate the need for MRD surveillance. However, WT1 expression in healthy PB and BM cells reduces the MRD analysis's sensitivity in AML patients. In comparison to the above-mentioned markers, the 9 NPM1 mutation is a more precise, sensitive, and durable molecular marker.

Complete remission rates were higher in AML with normal karyotype (AML-NK) patients with NPM1 mutant than in AML-NK patients without NPM1 mutant following induction treatment. NPM1 mutations independently predict indicators for chemotherapy response since the NPM1+ - FLT3- group only achieves greater rates of full remission.

The response rate is lowest in AML patients who have both mutations. A minimal residual disease assessment can forecast early recurrence and long-term survival. The prognosis is equally and significantly affected by the molecular analysis of NPM1 mutant transcript levels at two independent check points, the double induction therapy and complete consolidation therapy.[10,11] Two stages were taken to complete this investigation. We first established a quantitative test for measuring the NPM1 type A mutation, and then we tracked down AML patients who had this mutation at the time of diagnosis.

Fresh BM and/or PB samples were taken during follow-up from AML patients with NPMmut A at various times, including the moment of diagnosis (Ethical code: IR.TUMS.REC.1394.1296). Table 1 summarises the demographic and clinical features of the individuals that were enrolled. Ficoll gradient density separated mononuclear cells. Following the manufacturer's directions, cDNA was produced using Thermo Scientific Kit after total RNA was extracted using Roche's TriPure reagent. NPM1-containing cDNA In order to set up the NPM1 quantitative test, a mutation was run as a positive control and employed in the cloning procedure.

Negligible leftover infection (MRD) tests give early recognizable proof of hematologic backslide and ideal administration of intense myeloid leukemia (AML) patients.Around, half of AML patients don't have clonal chromosomal distortions and classify as a cytogenetically ordinary intense myeloid leukemia (CN-AML).[10]Sub-atomic markers are valuable in analyzation of this heterogeneous gathering of AML patients into prognostically various subgroups. Around 60% of grown-up CN-AML have a change in exon 12 of NPM1 quality.

This change is explicit for harmful clone furthermore, possibly is a decent marker of MRD more than different markers for example WT1 and FLT3-ITD. In this review study, we set up a NPM1 quantitative test and afterward, AML patients conveying NPM1mut were followed-up for MRD observing. In this review, we created RNA-based RQ-PCR to quantitation of NPM1 change A. Higher awareness of RNA recently was shown by Gorello P et al.

Opposite and forward unambiguous groundworks were utilized for enhancement of 12 exon in NPM1 quality conveying change An and test named with fluorescent colors (FAM and TAMRA), situated on exon intersection, was utilized for enhancement of cDNA as it were. 13 The outcomes showed that checking of NPM1 transformation A could be identified with responsiveness of 10-5.

This responsiveness is reasonable for MRD location. The responsiveness relies upon kinds of tests (PB, BM) furthermore, tests and it can shift between affirmative also, 10-6 In this review, backslide happened in 54.5 % of patients (n=6). All cases at determination exhibited something very similar change at backslide. Schnittger S et al. showed that the sort of NPM1 change was same in 84 matched conclusion backslide tests,10 and reasoned that this marker is steady in various period of sickness.All things considered, Kronke J et al. showed that NPMmut level was not recognizable in 5 of 45 patients at time of relapse.14 The new review results on a huge companion affirmed past outcomes as they found that NPM1 transformation was lost at backslide in 5/53 matched PB and/BM tests.

Variable NPMmut level with 3 logs or more contrasts at determination was accounted for by SchnittgerS et al.10 though in our review, the degree of articulation of NPMmut record at conclusion showed under 1 log contrast that was not huge that could be because of little examples size in this review.In early concentrate by Schnittger, the relationship between level of quality combinations at season of finding also, reaction to treatment was evaluated and showed that essential elevated degree of quality combinations was related with an unfriendly result in patients.Be that as it may, in their most far reaching concentrate on patients with NPM1 change, they revealed that NPMmut level at finding meaningfully affected any of the endurance boundaries.

In the current review, no critical relationship was found between freak record level and WBC furthermore, Plt count at analysis. Likewise, there was no critical relationship between the underlying degree of NPMmut and backslide. Because of little example size of,study, more examples are expected to endorse the end. Barragan E et al. surveyed MRD by WT1 what's more, NPM1 markers in 24 NPM1 type A-transformed AML patients, at the same time. There were 6 backslide tests of 5 patients and the degrees of the two markers at backslide were higher than at determination in PB and BM tests.Their outcomes were as per perceptions made by Schnittger S et al. who shown that NPM1mut levels at backslide were 2 logs higher than at determination in 84 matched examples of at determination/relapse.

In present review, in 1 of 6 patients with backslide,NPM1mut/ABL% level at first backslide was like at conclusion, which was expanded to 1.5 logs at second backslide. Likewise, the freak record level at backslide expanded to around 1 sign in 2 patients however in 3 last option cases it was lower than at finding. These results could be connected with assortment of tests at season of sub-atomic backslide and before clinical backslide.

In a review directed by Schnittger S et al. unique examples of treatment reaction were characterized in checking of MRD after first-line treatment and it was accounted for that patients who had basically a 5-log decrease in NPM1mut levels showed great reaction to treatment. In the current review, 6 patients experienced repeat whose NPM1mut decrease levels were under 5 logs later treatment. The patient with ITD−- NPM1+shown less forceful sickness contrasted and FLT3/inward pair duplication (ITD)+patient in third gathering.

Overall, despite the current research's constraints on sample size and follow-up time, this study amply confirms the reliability of the test method and illustrates how levels of mutant NPM1 decrease during induction therapy can have a significant impact on patient outcomes.

The Department of Hematology at Tehran University of Medical Sciences' School of Allied Medical Sciences provided financial support for the MSc thesis that led to this study. We appreciate the help in providing laboratory space, samples, and patient data from the Hematology-Oncology and Stem Cell Research Center, Shariati Hospital connected with Tehran University of Medical Sciences.

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Ali Ghasemi . Using the NPM1 Marker to Assess Minimal Residual Disease in Acute Myeloid Leukemia. World Journal Of Hematology And Oncology 2022.