Targeting NPM1 inhibits proliferation and promotes apoptosis of hepatic progenitor cells via suppression of mTOR signalling pathway.

BACKGROUND: Hepatic progenitor cells serve not only as the origin of combined hepatocellular cholangiocarcinoma (cHCC-CCA) but are also responsible for malignancy recurrence after surgical resection. Nucleophosmin 1 (NPM1) has been implicated in cancer metastasis and poor prognosis. This study aimed to determine the expression of NPM1 by hepatic progenitor cells in cHCC-CCA and the effects of targeting NPM1 on hepatic progenitor cells and BEL-7402 cells with characteristics of both progenitor cells and cHCC-CCA. METHODS: First, NPM1 was detected by RT‒PCR, western blotting, and double-immunofluorescence staining in cHCC-CCA tissues. NPM1 expression was subsequently analysed in rat hepatic progenitor cells cultured in vitro and in interleukin 6 (IL6)-treated cells. The effects and mechanism of NPM1 on hepatic progenitor cells were determined by knocking down NPM1 and performing RNA sequencing analysis. Finally, NSC348884, a small-molecule inhibitor that disrupts NPM1 dimer formation, was used to confirm the function of NPM1 in BEL-7402 cells. RESULTS: Both human hepatic progenitor cells in cHCC-CCA tissues and rat in vitro cultured hepatic progenitor cells highly expressed NPM1. IL6, a cytokine involved in the malignant transformation of hepatic progenitor cells, dose-dependently increased NPM1 and PCNA expression. Knocking down NPM1 reduced IL6R transcription (P < 0.0001) and inhibited the proliferation (P = 0.0065) of hepatic progenitor cells by suppressing the mTOR signalling pathway and activating the apoptosis pathway. Furthermore, knocking down NPM1 in hepatic progenitor cells resulted in more apoptotic cells (7.33 ± 0.09% vs. 3.76 ± 0.13%, P < 0.0001) but fewer apoptotic cells in the presence of NSC348884 (47.57 ± 0.49% vs. 63.40 ± 0.05%, P = 0.0008) than in the control cells, suggesting that low-NPM1-expressing cells are more resistant to NSC348884. In addition, NSC348884 induced the apoptosis of BEL-7402 cells with an IC50 of 2.77 µmol/L via the downregulation of the IL-6R and mTOR signalling pathways and inhibited the growth of BEL-7402 cells in a subcutaneous xenograft tumour model (P = 0.0457). CONCLUSIONS: Targeting NPM1 inhibits proliferation and induces apoptosis in hepatic progenitor cells and BEL-7402 cells, thus serving as a potential therapy for cHCC-CCA.

Characterization of the AGR2-NPM3 axis uncovers the AGR2 involvement in PD-L1 regulation in colorectal cancer.

Despite extensive research, the molecular role of AGR2 in the progression and metastasis of colorectal cancer (CRC) has not been fully characterized. We used quantitative mass spectrometry (SWATH MS) to identify differentially expressed proteins in paired CRC cell models of the SW480 and SW620 cell lines in response to AGR2 protein level manipulation. Relying on the results from SWATH MS and subsequent immunochemical validation, we selected NMP3 as the top candidate protein associated with AGR2 in CRC tumour cells in our screen. RT‒qPCR and immunochemical analysis confirmed the involvement of AGR2-mediated regulation of NPM3 at the transcriptional and posttranscriptional levels. Since PD-L1 is a constituent of the NPM3 regulatory axis, we aimed to correlate the changes in PD-L1 to the differential expression of AGR2 in our cell models. We found that AGR2 positively regulates PD-L1 levels in both SW480 and SW620 cell lines; additionally, several different CRC patient transcriptome cohorts confirmed the association of AGR2 with PD-L1. Our work reveals a new AGR2-NPM3 regulatory axis and the involvement of AGR2 in the regulation of PD-L1, which paves the way for the association of AGR2 with immune evasion in CRC cells.

Early Prediction and Streamline of Nucleophosmin Mutation Status in Acute Myeloid Leukemia Using Cup-Like Nuclear Morphology.

Background and Objectives: With the advent of novel therapies for nucleophosmin gene (NPM1)-mutated acute myeloid leukemia (AML), there is a growing need for the reliable prediction of NPM1 mutations. This study explored the role of cytomorphological features in the early prediction of NPM1-mutated AML. Materials and Methods: Altogether, 212 de novo AML cases with normal karyotypes, diagnosed and treated at a single institution within 5 years (2018-2023), were retrospectively evaluated. A final diagnosis of NPM1-mutated AML, based on the World Health Organization (WHO) integrated criteria, including real-time based identification of NPM1 mutation and normal karyotype, was established in 83/212 (39.15%) cases. Results: Cup-like blasts (CLBs), a cytomorphological feature suggestive of NPM1-mutated AML, were detected in 56/83 (67%) patients. Most cases (44/56, 78.6%) had CLB ≥ 10%. In total, 27 of 83 AML NPM1-mutated patients had no CLB morphology (missed call). Additionally, two of 212 had CLB morphology without confirmed NPM1 mutation (wrong call). The positive/negative predictive values of cytomorphological evaluation for CLB ≥ 10% were 95.7%/75.6%, with sensitivity/specificity of 53%/98.5%, while the accuracy was 80.7%. We noted an increased percentage of CLBs (≥15%) in 77.8% and 50% of patients with AML without and with granulocytic maturation, respectively (the specificity for NPM1 mutation prediction was 100%). CLB was associated with fms-like tyrosine kinase 3 (FLT3) mutation (p = 0.03), but, without statistical significance for CLB ≥ 10% and CLB ≥ 15%. Conclusions: Our investigation confirmed that the morphological identification of CLB at diagnosis represents a reliable and easily reproducible tool for the early prediction of NPM1 mutations, enabling a streamlined genetic work-up for its confirmation. This may facilitate considering the early administration of individualized therapies by clinicians for specific patients.

Evolution from an antecedent chronic myeloid malignancy does not impact survival outcomes in NPM1-mutated AML.

Nucleophosmin-1 (NPM1)-mutated AML is a molecularly defined subtype typically associated with favorable treatment response and prognosis; however, its prognostic significance in AML evolving from an antecedent chronic myeloid malignancy is unknown. This study's primary objective was to determine the impact of mutated NPM1 on the prognosis of AML evolving from an antecedent chronic myeloid malignancy. We conducted a retrospective chart review including patients with NPM1-mutated de novo and sAML. sAML was defined as those with a preceding chronic-phase myeloid malignancy before diagnosis of AML. Of 575 NPM1-mutated patients eligible for inclusion in our study, 51 (8.9%) patients were considered to have sAML. The median time from diagnosis of NPM1-mutated chronic myeloid malignancy to sAML evolution was 3.6 months (0.5-79.3 months). No significant differences in leukemia-free (2-year LKFS 52.0% vs. 51.2%, p = .9922) or overall survival (2-year OS 56.3% vs. 49.4%, p = .4246) were observed between patients with NPM1-mutated de novo versus sAML. Our study suggests that evolution from a preceding myeloid malignancy is not a significant predictor of poor prognosis in the setting of an NPM1 mutation. Our study demonstrated a short time to progression to sAML in most patients, which further supports the consideration of NPM1 as an AML-defining mutation.

Electrostimulation Evokes Caspase-3-Activated Fast Cancer Cell Pyroptosis and Its Nuclear Stress Response Pathways.

Pyroptosis of programmed cell death has been recognized as a more effective way to inhibit the occurrence and development of tumors than the better-studied apoptosis. However, it is still challenging to quickly and effectively trigger pyroptosis of cancer cells for high-efficacy cancer treatment. Here, we report on the first use of mild constant-potential electrostimulation (cp-ES) to quickly trigger cancer cell pyroptosis with a probability up to ∼91.4% and significantly shortened time (within 1 h), ∼3-6 times faster than typical drug stimulation to induce pyroptosis. We find that the ES-induced cancer cell pyroptosis is through the activated caspase-3 (pathway) cleavage of gasdermin E (GSDME) to form an N-terminal fragment (GSDME-N) and observe nuclear shrinkage and reduction of the number of nucleoli as well as down-/up-regulated expression of two important nucleoproteins of nucleolin and nucleophosmin (NPM1). The study enriches the basic understanding of pyroptosis and provides a new avenue for potential effective treatment of cancer.

Menin inhibitors in pediatric acute leukemia: a comprehensive review and recommendations to accelerate progress in collaboration with adult leukemia and the international community.

Aberrant expression of HOX and MEIS1 family genes, as seen in KMT2A-rearranged, NUP98-rearranged, or NPM1-mutated leukemias leads to arrested differentiation and leukemia development. HOX family genes are essential gatekeepers of physiologic hematopoiesis, and their expression is regulated by the interaction between KMT2A and menin. Menin inhibitors block this interaction, downregulate the abnormal expression of MEIS1 and other transcription factors and thereby release the differentiation block. Menin inhibitors show significant clinical efficacy against KMT2A-rearranged and NPM1-mutated acute leukemias, with promising potential to address unmet needs in various pediatric leukemia subtypes. In this collaborative initiative, pediatric and adult hematologists/oncologists, and stem cell transplant physicians have united their expertise to explore the potential of menin inhibitors in pediatric leukemia treatment internationally. Our efforts aim to provide a comprehensive clinical overview of menin inhibitors, integrating preclinical evidence and insights from ongoing global clinical trials. Additionally, we propose future international, inclusive, and efficient clinical trial designs, integrating pediatric populations in adult trials, to ensure broad access to this promising therapy for all children and adolescents with menin-dependent leukemias.

Common Driver Mutations in AML: Biological Impact, Clinical Considerations, and Treatment Strategies.

Next-generation sequencing of samples from patients with acute myeloid leukemia (AML) has revealed several driver gene mutations in adult AML. However, unlike other cancers, AML is defined by relatively few mutations per patient, with a median of 4-5 depending on subtype. In this review, we will discuss the most common driver genes found in patients with AML and focus on the most clinically relevant ones that impact treatment strategies. The most common driver gene mutations in AML occur in NPM1 and FLT3, accounting for ~30% each. There are now targeted therapies being tested or already approved for these driver genes. Menin inhibitors, a novel targeted therapy that blocks the function of the menin protein, are in clinical trials for NPM1 driver gene mutant AML after relapse. A number of FLT3 inhibitors are now approved for FLT3 driver gene mutant AML in combination with chemotherapy in the frontline and also as single agent in relapse. Although mutations in IDH1/2 and TP53 only occur in around 10-20% of patients with AML each, they can affect the treatment strategy due to their association with prognosis and availability of targeted agents. While the impact of other driver gene mutations in AML is recognized, there is a lack of data on the actionable impact of those mutations.

Nucleophosmin 1 promotes mucosal immunity by supporting mitochondrial oxidative phosphorylation and ILC3 activity.

Nucleophosmin 1 (NPM1) is commonly mutated in myelodysplastic syndrome (MDS) and acute myeloid leukemia. Concurrent inflammatory bowel diseases (IBD) and MDS are common, indicating a close relationship between IBD and MDS. Here we examined the function of NPM1 in IBD and colitis-associated colorectal cancer (CAC). NPM1 expression was reduced in patients with IBD. Npm1+/- mice were more susceptible to acute colitis and experimentally induced CAC than littermate controls. Npm1 deficiency impaired the function of interleukin-22 (IL-22)-producing group three innate lymphoid cells (ILC3s). Mice lacking Npm1 in ILC3s exhibited decreased IL-22 production and accelerated development of colitis. NPM1 was important for mitochondrial biogenesis and metabolism by oxidative phosphorylation in ILC3s. Further experiments revealed that NPM1 cooperates with p65 to promote mitochondrial transcription factor A (TFAM) transcription in ILC3s. Overexpression of Npm1 in mice enhanced ILC3 function and reduced the severity of dextran sulfate sodium-induced colitis. Thus, our findings indicate that NPM1 in ILC3s protects against IBD by regulating mitochondrial metabolism through a p65-TFAM axis.

Caspase-2 is essential for proliferation and self-renewal of nucleophosmin-mutated acute myeloid leukemia.

Mutation in nucleophosmin (NPM1) causes relocalization of this normally nucleolar protein to the cytoplasm (NPM1c+). Despite NPM1 mutation being the most common driver mutation in cytogenetically normal adult acute myeloid leukemia (AML), the mechanisms of NPM1c+-induced leukemogenesis remain unclear. Caspase-2 is a proapoptotic protein activated by NPM1 in the nucleolus. Here, we show that caspase-2 is also activated by NPM1c+ in the cytoplasm and DNA damage-induced apoptosis is caspase-2 dependent in NPM1c+ but not in NPM1wt AML cells. Strikingly, in NPM1c+ cells, caspase-2 loss results in profound cell cycle arrest, differentiation, and down-regulation of stem cell pathways that regulate pluripotency including impairment of the AKT/mTORC1 pathways, and inhibition of Rictor cleavage. In contrast, there were minimal differences in proliferation, differentiation, or the transcriptional profile of NPM1wt cells lacking caspase-2. Our results show that caspase-2 is essential for proliferation and self-renewal of AML cells expressing mutated NPM1. This study demonstrates that caspase-2 is a major effector of NPM1c+ function.

Nucleophosmin: A Nucleolar Phosphoprotein Orchestrating Cellular Stress Responses.

Nucleophosmin (NPM1) is a key nucleolar protein released from the nucleolus in response to stress stimuli. NPM1 functions as a stress regulator with nucleic acid and protein chaperone activities, rapidly shuttling between the nucleus and cytoplasm. NPM1 is ubiquitously expressed in tissues and can be found in the nucleolus, nucleoplasm, cytoplasm, and extracellular environment. It plays a central role in various biological processes such as ribosome biogenesis, cell cycle regulation, cell proliferation, DNA damage repair, and apoptosis. In addition, it is highly expressed in cancer cells and solid tumors, and its mutation is a major cause of acute myeloid leukemia (AML). This review focuses on NPM1's structural features, functional diversity, subcellular distribution, and role in stress modulation.

[Appropriate use of the ELN guidelines based on results of real-world genetic analysis in Japanese patients with AML].

Researchers in the field of acute myeloid leukemia have long sought to establish a prognostic stratification system for clinical use that combines multiple genetic mutations. In 2022, the European LeukemiaNet (ELN) proposed a new prognostic model incorporating new genetic mutations. However, Japanese National Health insurance only recently began covering clinical genetic analysis for AML. We established the Multi-center Collaborative Program for Gene Sequencing of Japanese AML (GS-JAML) to contribute to clinical practice by providing rapid genetic analysis results. Retrospective analysis of this research program revealed (1) the clinical significance of CEBPA-bZIP mutations, and (2) the clinical significance of DNMT3A mutations in NPM1 mutated AML.

Influence of genetic co-mutation on chemotherapeutic outcome in NPM1-mutated and FLT3-ITD wild-type AML patients.

BACKGROUND: Nucleophosmin 1 (NPM1) gene-mutated acute myeloid leukemia (NPM1mut AML) is classified as a subtype with a favorable prognosis. However, some patients fail to achieve a complete remission or relapse after intensified chemotherapy. Genetic abnormalities in concomitant mutations contribute to heterogeneous prognosis of NPM1mut AML patients. METHODS: In this study, 91 NPM1-mutated and FLT3-ITD wild-type (NPM1mut/FLT3-ITDwt) AML patients with intermediate-risk karyotype were enrolled to analyze the impact of common genetic co-mutations on chemotherapeutic outcome. RESULTS: Our data revealed that TET1/2 (52/91, 57.1%) was the most prevalent co-mutation in NPM1mut AML patients, followed by IDH1/2 (36/91, 39.6%), DNMT3A (35/91, 38.5%), myelodysplastic syndrome related genes (MDS-related genes) (ASXL1, BCOR, EZH2, RUNX1, SF3B1, SRSF2, STAG2, U2AF1 and ZRSR2 genes) (35/91, 38.5%), FLT3-TKD (27/91, 29.7%) and GATA2 (13/91, 14.3%) mutations. Patients with TET1/2mut exhibited significantly worse relapse-free survival (RFS) (median, 28.7 vs. not reached (NR) months; p = 0.0382) compared to patients with TET1/2wt, while no significant difference was observed in overall survival (OS) (median, NR vs. NR; p = 0.3035). GATA2mut subtype was associated with inferior OS (median, 28 vs. NR months; p < 0.0010) and RFS (median, 24 vs. NR months; p = 0.0224) compared to GATA2wt. By multivariate analysis, GATA2mut and MDS-related genesmut were independently associated with worse survival. CONCLUSION: Mutations in TET1/2, GATA2 and MDS-related genes were found to significantly influence the chemotherapeutic outcome of patients with NPM1mut AML. The findings of our study have significant clinical implications for identifying patients who have an adverse response to frontline chemotherapy and provide a novel reference for further prognostic stratification of NPM1mut/FLT3-ITDwt AML patients.

[Clinical Characteristics and Prognostic Relevance of Co-Mutated Genes in Acute Myeloid Leukemia Patients with FLT3 Mutations].

OBJECTIVE: To investigate the clinical characteristics and influence of co-mutated gene on acute myeloid leukemia patients (AML) with FMS-like tyrosine kinase-3 (FLT3) mutations. METHODS: A total of 273 FLT3+ AML patients were enrolled, and the co-mutation gene data of the patients were collected to further analyze the prognosis of the patients. FLT3 and other common mutations were quantified by PCR amplification products direct sequencing and second-generation sequencing (NGS). RESULTS: When patients were divided into FLT3- ITD +, FLT3- TKD +, FLT3- ITD ++TKD + and FLT3- ITD -+TKD - group according to the type of FLT3 mutations, it was found that the frequencies of TET2, GATA2, NRAS and ASXL1 mutation were significantly different among the 4 groups (all P < 0.05). When patients were divided into allelic ratio (AR) ≥0.5 and <0.5 group, it was found that the frequencies of FLT3- ITD +, FLT3 -ITD - +TKD -, NPM1, NRAS and C-kit were significantly different between the two groups (all P < 0.05). When patients were divided into normal and abnormal karyotype group, it was found that the frequencies of FLT3- ITD +, FLT3- TKD +, NPM1, GATA2 and C-kit were significantly different between the two groups (all P < 0.05). The median overall survival (OS) of AML patients with FLT3 -TKD + (including FLT3- ITD ++TKD +) was longer than that of patients with FLT3- ITD + alone (P < 0.05). The OS and relapse-free survival (RFS) of AML patients with FLT3++TET2+ were both shorter than those of patients with FLT3++TET2- (both P < 0.05). CONCLUSION: The mutation frequencies of co-mutated genes are correlated with subtypes of FLT3, karyotype and AR. AML patients with FLT3 -TKD + have longer OS than patients with FLT3- ITD + alone, and patients with co-mutation of TET2 have shorter median OS and RFS.

[Characteristic Analysis of Adult Acute Myeloid Leukemia Patients with PTPN11 Gene Mutation].

OBJECTIVE: To investigate the incidence of PTPN11 gene mutation and its associated gene mutations in adult patients with acute myeloid leukemia (AML), and analyze its clinical characteristics. METHODS: Second-generation sequencing and Sanger sequencing were used to detect 51 gene mutations, and multiplex-PCR was used to detect 41 fusion genes from 451 newly diagnosed adult AML patients admitted to Affiliated Hospital of Jiangnan University, Changzhou Second People's Hospital, Wuxi People's Hospital and Wuxi Second People's Hospital from January 2017 to July 2022. RESULTS: Among 451 primary adult AML patients, the PTPN11 gene mutation was detected in 34 cases, and the mutation rate was 7.5%. In the 34 patients, 37 PTPN11 alterations were found, which were exclusively missense mutations affecting residues located within the N-SH2 (31 cases) and PTP (6 cases) domains and clustered overwhelmingly in exon 3. The platelet count of PTPN11 mutation patients was 76.5(23.5, 119.0)×109/L, which was significantly higher than 41.0(22.0, 82.5)×109/L of wild-type patients (P < 0.05). While, there were no significant differences in sex, age, peripheral white blood cell count, hemoglobin, and bone marrow blast between PTPN11 mutation and wild-type patients (P >0.05). In FAB subtypes, PTPN11 mutations were mainly distributed in M5, followed by M2 and M4, less frequently in M3 and M6. There was no significant difference in the distribution of FAB subtypes between PTPN11 mutation and wild-type patients (P >0.05). A total of 118 AML patients were detected positive fusion gene, among which patients with PTPN11 mutations had a higher incidence of positive MLL-AF6 than wild-type ones (P < 0.01). 97.1% of 34 patients with PTPN11 mutations were accompanied by other mutations, in descending order, they were respectively NPM1 (38.2%), NRAS (32.4%), FLT3-ITD (32.4%), DNMT3A (32.4%) and KRAS (23.5%), etc . CONCLUSION: PTPN11 mutation has a certain incidence in AML patients and is clustered overwhelmingly in exon 3. ALL of them are exclusively missense mutations, and most often present in conjunction with NPM1 mutations. FAB typing of PTPN11 mutation is mostly manifested as M5 subtype, which is associated with higher platelet counts.

Molecular relapse after first-line intensive therapy in patients with CBF or NPM1-mutated acute myeloid leukemia - a FILO study.

Patients with Core-Binding Factor (CBF) and NPM1-mutated acute myeloid leukemia (AML) can be monitored by quantitative PCR after having achieved first complete remission (CR) to detect morphologic relapse and drive preemptive therapy. How to best manage these patients is unknown. We retrospectively analyzed 303 patients with CBF and NPM1-mutated AML, aged 18-60 years, without allogeneic hematopoietic cell transplantation (HCT) in first CR, with molecular monitoring after first-line intensive therapy. Among these patients, 153 (51%) never relapsed, 95 (31%) had molecular relapse (53 received preemptive therapy and 42 progressed to morphologic relapse at salvage therapy), and 55 (18%) had upfront morphologic relapse. Patients who received preemptive therapy had higher OS than those who received salvage therapy after having progressed from molecular to morphologic relapse and those with upfront morphologic relapse (three-year OS: 78% vs. 51% vs. 51%, respectively, P = 0.01). Preemptive therapy included upfront allogeneic HCT (n = 19), intensive chemotherapy (n = 21), and non-intensive therapy (n = 13; three-year OS: 92% vs. 79% vs. 58%, respectively, P = 0.09). Although not definitive due to the non-randomized allocation of patients to different treatment strategies at relapse, our study suggests that molecular monitoring should be considered during follow-up to start preemptive therapy before overt morphologic relapse.

An unusual case of pure erythroid leukemia with normal karyotype and NPM1 mutation.

Pure erythroid leukemia (PEL) is an extremely rare subtype of acute myeloid leukemia (AML). Although not specific, PEL is almost uniformly associated with complex karyotype and TP53 mutations. Given the rarity of the disease, our understanding of its cytogenetic and molecular features deems incomplete. We aim to complement existing literature by presenting an unusual case of PEL. The case is comprehensively worked up with multiple modalities. We present for the first time a case of PEL with unusual cytogenetic and molecular features: normal karyotype with absence of TP53 mutations and presence of NPM1 and NRAS mutations. This is a valuable addition to literature, expanding our understanding of molecular and cytogenetic spectra of PEL.

HOXA9 Regulome and Pharmacological Interventions in Leukemia.

HOXA9, an important transcription factor (TF) in hematopoiesis, is aberrantly expressed in numerous cases of both acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) and is a strong indicator of poor prognosis in patients. HOXA9 is a proto-oncogene which is both sufficient and necessary for leukemia transformation. HOXA9 expression in leukemia correlates with patient survival outcomes and response to therapy. Chromosomal transformations (such as NUP98-HOXA9), mutations, epigenetic dysregulation (e.g., MLL- MENIN -LEDGF complex or DOT1L/KMT4), transcription factors (such as USF1/USF2), and noncoding RNA (such as HOTTIP and HOTAIR) regulate HOXA9 mRNA and protein during leukemia. HOXA9 regulates survival, self-renewal, and progenitor cell cycle through several of its downstream target TFs including LMO2, antiapoptotic BCL2, SOX4, and receptor tyrosine kinase FLT3 and STAT5. This dynamic and multilayered HOXA9 regulome provides new therapeutic opportunities, including inhibitors targeting DOT1L/KMT4, MENIN, NPM1, and ENL proteins. Recent findings also suggest that HOXA9 maintains leukemia by actively repressing myeloid differentiation genes. This chapter summarizes the recent advances understanding biochemical mechanisms underlying HOXA9-mediated leukemogenesis, the clinical significance of its abnormal expression, and pharmacological approaches to treat HOXA9-driven leukemia.

Refinement of Risk-Stratification of Cytogenetically Normal Acute Myeloid Leukemia Adult Patients by MN1 Expression.

INTRODUCTION: Acute myeloid leukemia with normal cytogenetics (CN-AML) represents a heterogeneous group having diverse genetic mutations. Understanding the significance of each of these mutations is necessary. In this study, we evaluated the prognostic role of MN1 expression in adult CN-AML patients. METHOD: One hundred and sixty-three de-novo adult AML patients were evaluated for MN1 expression by real-time PCR. MN1 expression was correlated with the clinical characteristics of the patients and their outcomes. RESULTS: Higher MN1 expression was associated with NPM1 wild-type (p<0.0001), CD34 positivity (p=0.006), and lower clinical remission rate (p=0.027). FLT3-ITD and CEBPA mutations had no association with MN1 expression. On survival analysis, a high MN1 expression was associated with poor event-free survival (Hazard Ratio 2.47, 95% Confidence Interval: 1.42-4.3; p<0.0001) and overall survival (Hazard Ratio 4.18, 95% Confidence Interval: 2.17-8.08; p<0.0001). On multivariate analysis, the MN1 copy number emerged as an independent predictor of EFS (p<0.0001) and OS (p<0.0001). CONCLUSION: MN1 expression is an independent predictor of outcome in CN-AML.