Prognostic Involvement of Nucleophosmin Mutations in Acute Myeloid

In World Health Organization 2008, AML with mutated NPM have assigned a separate class of myeloid nucleoplasm which have unique molecular, prognostic and pathological feature (Michael et al., 2010). Various authors attribute mutated NPM as favorable prognostic marker for Overall survival or event free survival in AML having AML_NK. This attribution makes NPM an important candidate to study for the better understanding of leukemogenesis.


Introduction
In World Health Organization 2008, AML with mutated NPM have assigned a separate class of myeloid nucleoplasm which have unique molecular, prognostic and pathological feature (Michael et al., 2010).Various authors attribute mutated NPM as favorable prognostic marker for Overall survival or event free survival in AML having AML_NK.This attribution makes NPM an important candidate to study for the better understanding of leukemogenesis.

Structure of NPM Gene and Protein
Human nucleophosmin (NPM1) gene, is located at chromosome 5q35 and comprises of 12 exons (Chang et al., 1990).It is a phosphoprotein mainly present in nucleolus granular region, but shuttles continuously between nucleus and cytoplasm (Fallani et al., 2005).This shuttling is important in biogenesis of ribosome as well as in the transportation of preribosomal particles while in cytoplasm it binds to the unduplicated centrosome and regulates cell division (Fallini et al., 2005).NPM stabilizes genome via regulation of DNA repair process (Lee et al., 2005).
The nonpolar N-terminus region and multimeric region of NPM is essential and directly involved in the correct assemblage of maturing ribosomes in the nucleolus (Hingorani et al., 2000).The middle portion of NPM contains 2 acidic stretches which helps in histone binding (Ouwaki et al., 2001) while the in between fragment of the acidic stretches pertain ribonuclease activity.The C-terminus domain, binds with nucleic acid also have ribonuclease activity and is followed by short aromatic stretch which is critical for NPM binding to nucleolus (Hingorani et al., 2000).
NPM inhibits DNA fragmentation activity and plays a crucial role in hematopoietic stem cell modulation, regulation of DNA integrity and tumor suppressors genes p53 and ARF.NPM is undoubtedly important for balanced cell growth but the beneficial effects of NPM decreases upon maturity.The overexpression of NPM enhances chances of survival and recovery of hematopoietic stem cells under stress conditions on one hand while on other hand (Li et al., 2006) it promotes abnormal cell growth in malignant cells.

Interaction with p53
NPM regulates p53 levels and activity.There is a close association between NPM, "nucleolar integrity" and p53 stability (Colombo et al., 2002) nucleolus behaves as a stress sensor where NPM plays an important role to arrest "p53 dependent cell cycle" (Kurki et al., 2004).

Interaction with ARF
NPM and ARF both mostly localize in the nucleolus.(Bertwistle et al., 2004).They interact in a mutually beneficial way.Hence, NPM prevent ARF from destruction while ARF control NPM polyubiquitination (Kuo et al 2004;Grisendi et al., 2005).Although this interactions is still in debate but under cellular stress condition, NPM and ARF are reorganized to the nucleoplasm (Gjerset et al., 2006).
NPM1 mutations are more stable than FLT3 mutations and contrary to FLT3 mutations,they are also present at relapse while NPM negative patients at presentation cannot acquired them at relapse or during the malignancy which indicates that NPM mutations are not directly involved in advancement of disease.Lin et al. (2006) reported the mutually exclusive nature of NPM and CEBPA.In some of the cases, the loss of NPM1 mutations was typically responsible for the transformation of normal karyotype into abnormal karyotype (Suzuki et al., 2005;Chou et al., 2006).

Discovery and various types of NPM1 mutations in AML
The first attempt to detect NPM mutation was done by GIMEMA/AML12 EORTC trial.This trial screened 591 AML-NK patients and observed cytoplasmic NPM (NPMc).The sequencing of above mentioned cases confirmed mutations in exon 12.After this, no of studies confirmed that Frame shift mutations in exon 12 result in loss of a nucleolar localization signal and halt movement of mutant protein to the cytoplasm (Brown et al., 2007;Fallini et al., 2008 ;Kim et al., 2010;Matson et al, 2010;Kaseem et al., 2011).More than 40 different NPM1 mutations in exon 12 have been identified in AML and all are highly constrained to exon 12, except two mutations which involves exon 9 and exon 11 (Albiero et al., 2006;Mariona et al., 2006).In majority, NPM mutations harbor type A mutations (75-80%) while type B and type D comprises (10%), and (5%) mutations respectively (Falini et al., 2007;Szankasi et al., 2008;Kaseem et al., 2011).These four mutations represent about 90-95% of all NPM1-positive cases (Hafeez et al., 2010) and 60% of them have normal karyotype AML (Brown et al., 2007).Table 1 has shown recent information about various types of NPM mutation.

Role of NPM mutation in promoting leukemia
NPM mutations are rarely seen with chromosome abnormalities and are more common in normal kayotypes, this is the indication that they play preliminary role in the process of leukemogenesis (Colombo et al., 2005;2006).However, it is still unclear that how the mutant protein propagates leukemia.Uptill now various studies reported that all NPM mutations result in abnormal dislocalization of the mutant protein into the cytoplasm which causes "leukemogenesis" (Bolli et al., 2007;Falini et al., 2009).Accelerated transport of nucleophosmin into cytoplasm probably triggers "multiple cellular pathways" by "loss of function"/or "gain of function".It also causes the interaction of NPM mutant protein to others proteins which are present in cytoplasm.Although no study still confirms that this interaction causes "leukemogenesis" but it was reported that mutant protein involved in "knock down the oncosuppressor ARF" (denBesten et al., 2005;Colombo et al., 2006) and activates c-MYC oncogene (Bonneti et al., 2008).Further more, dislocation of mutant protein causes lessen amount of wild-type NPM1 in the nucleolus which result in dislocation into cytoplasm by the formation of heterodimers with NPM1 mutant..and loss of heterozygosity.NPM heterozygous cells are more susceptible to "oncogenic transformation" (Falini et al., 2009).One study in NPM knockout mouse reported that, NPM inactivation creates genomic instability which, promotes cancer susceptibility in vitro and in vivo (Falini et al., 2011).

Conclusion
NPM mutations are presently the most prevailing mutations in AML-NK.In absence of other genetic abnormalies they have proven their crucial prognostic importance in intermediate risk group.These mutations provide favorable response and better overall survival in absence of Internal Tendom Duplication (ITD) in AML patients.Hence ITD-/NPM+ genotype have shown over all better response in AML_NK.

20.3 10.1 6.3 51.1 30.0 31.3 54.2 46.8 56.3 33.1 30.0 31.3 23.7 38.0 31.3 Table 1. Prognostic Importance/Clinical Out Come of NPM1 Mutations in AML S Name of Author No of NPM cases ITD status Type of NPM mutation/ Insertion at nucleotide Prognosis comments 1
NPM1mut patients had significantly better CR rate than NPM1wt patients.NPM-1mut patients also had a significantly longer OS compared with NPM1wt patients 10 No difference in survival was observed among NPM1-mutated AML patients independently of whether they carried a NK or an AK, the NPM1-mutated/ FLT3-ITD negative cases showing the better prognosis EFS was significantly shorter in the NPM1-mutated/FLT3-ITD_ subgroup versus NPM1-mutated/ FLT3-ITD_FLT3-ITD negative cases, no statistically significant difference emerged in OS and EFS of NPM1-mutated 12