Somatic mutations of isocitrate dehydrogenases 1 and 2 are prognostic and follow-up markers in patients with acute myeloid leukaemia with normal karyotype

Patients with acute myeloid leukaemia with normal karyotype (AML-NK) comprise 40-50% of all AML patients.¹ They are characterized by high heterogeneity in terms of clinical features, biological characteristics and response to treatment. Nevertheless, all of the AML-NK patients are categorized into intermediate risk group. The need for more precise risk stratification of such cases led to the discovery of numerous new molecular markers. Some of them, such as mutations in fms-related tyrosine kinase-3 (FLT3), nucleophosmin (NPM1) and CCAAT/enhancer binding protein alpha (CEBPA) genes have made an impact on prognosis of AML-NK patients. Those mutations have been already included in the revised version of World Health Organisation classification of leukaemia.² This new classification implies that all AML-NK patients with mutated NPM1 without FLT3- (internal tandem duplication)ITD and mutated CEBPA have favourable genotype.

Mardis et al. have reported the entire genome sequence of leukemic cells from a single de novo AML-NK patient and compared it with the genome sequence from normal skin cells of the same patient.³ After that, from the number of possible somatic mutations, only a handfull of genes were recurrently mutated in multiple AML genomes, including mutations in the genes for isocitrate dehydrogenase 1 (IDH1) and isocitrate dehydrogenase 2 (IDH2).

The IDH1 and IDH2 genes, located at chromosome bands 2q33.3 and 15q26.1 respectively, encode NADPH (reduced nicotinamide adenine dinucleotide phosphate) - dependent isocitrate dehydrogenase 1/2 enzymes, whose main role is to protect cells from oxidative stress.⁴

Heterozygous point mutations in IDH1 and IDH2 genes most likely affect the evolutionarily conserved arginine at position R132 in exon 4 of IDH1 (IDH^R132) and either the homologous position R172 (IDH^R172) or the second arginine R140 in the IDH2 gene (IDH^R140).⁵

IDH1 and IDH2 mutations occur in approximately 20% of AML-NK cases.^6–11 Clinical characteristics commonly found in these patients compared to those with wild-type IDH are older age, higher platelet counts and concurrent presence of NPM1 mutations.^{5, 6, 8, 9, 11, 18} The relatively high incidence of IDH mutations and their association with the most commonly detected mutations in AML patients (NPM1 mutations) indicates possible mutual interactions in the pathogenesis of the disease.^{19, 22}

Despite the results of numerous studies investigating the effect of the presence of IDH mutations on clinical outcome, the prognostic significance of these mutations remains controversial.¹¹ A number of studies showed that the presence of these mutations have no effect in response to therapy and survival^{5, 14–16}, while there are others that suggest a negative prognostic effect.^{8–10, 17–20} Nevertheless, most studies agree with the fact that IDH mutations have adverse prognostic impact in the so called low-risk group of patients (NPM1⁺/FLT3-ITD^- AML-NK patients).^{8–10, 13, 20, 21}

Some studies investigated the potential of IDH mutations as a follow-up markers.^{13, 16, 22–24}IDH1 and IDH2 mutations are relatively stable and show direct correlation with disease status. Thus, IDH mutations could be useful markers for monitoring disease, including treatment response, minimal residual disease (MRD), and early relapse.

The purpose of our study was to analyze the frequency of mutations in IDH1/2 genes and their potential associations with other prognostic markers and outcome in 110 adult de novo AML-NK patients. We also analyzed the stability of these mutations during the course of the disease in complete remission (CR) and relapse.

The frequency of IDH mutations in patients with AML is 6-19%, but 12-33% in those with AMLNK.^{5, 8, 17, 18, 20, 33} In our study on adult AML-NK patients, IDH mutations were detected in 23% of them. The prevalence of IDH2 over IDH1 mutations observed here (15.5% vs 7%) was similar to other published results.^{5, 6, 8, 22}

Our patients with IDH mutations had higher platelet counts and a higher percentage of PB blasts than those without such mutations, which confirms previous findings.^{5, 6, 8, 11} We detected IDH mutations most frequently in M2 type cases, followed by M1 (36% and 24%, respectively) and M4 type, which is in according with other results.^{13, 15, 19}

Examining correlations between IDH mutations and other common genetic alterations in AML, such as NPM1 and FLT3 mutations, we found a slight but non-significant prevalence of NPM1⁺ among IDH⁺ patients (NPM1⁺- 26.2% vs NPM1^--20.6%; p = 0.496). This is not in line with previous reports.^{6, 8, 9, 11, 18} The FLT3 mutations were almost equally distributed between IDH⁺ and IDH^- groups of patients, which is in concordance with other studies.^{5, 7, 18}

The prognostic impact of IDH mutation is controversial. Most studies have shown that both IDH1 and IDH2 mutations confer an unfavourable prognosis in AML-NK, i.e. a higher risk of disease relapse and shorter OS.^{3, 6–8, 11, 16, 17, 20} In our study, CR rate was 62.2% in IDH^- patients, while in IDH⁺ patients it was somewhat lower (45.8%), but without statistical significance. A similar finding was reported by Nomdedeu et al.¹⁷, where the CR rate of IDH^- patients was 80% and 63% in IDH⁺ (p = 0.086).

We were able to demonstrate that IDH mutations act as an adverse prognostic marker of OS in AML-NK patients. That is, patients with IDH mutations had significantly worse OS, with a tendency for shorter DFS. This also confirmed earlier findings.^{6–8, 11, 16, 17, 20} Among the IDH⁺ patients, OS rate in those who received allogeneic SCT was significantly higher than that in those not given it. This was also observed by Yamaguchi et al.¹¹ and suggests that allogeneic SCT may improve OS in younger patients with IDH mutations.

The emergence of new molecular markers in AML-NK has contributed to a better and more precise classification of patients. This group is identified as an intermediate risk group, but because of its heterogeneity in terms of clinical outcome of the disease, more precise allocation is necessary. In addition to the FLT3 and NPM1 gene mutations that have already found significance as valuable prognostic factors, the detection of IDH mutations has contributed to refined risk classification of AMLNK patients.

When we applied molecular classification based on the presence/absence of NPM1 and FLT3 mutations in our cohort of patients, we observed that the presence of IDH mutations had an adverse impact on OS in the intermediate risk subgroup (NPM1^-/FLT3-ITD^-). This finding, already reported by others^{11, 16}, argues in favour of testing for IDH mutations among AML-NK patients.

The frequent co-occurrence of IDH mutations with NPM1 and less often with FLT3 mutations, indicates that such mutations cooperate in the process of leukemogenesis. IDH1 and IDH2 are epigenetic modifier genes involved in DNA methylation and histone modification, and do not completely fit into our current definition of type-I and type-II aberrations, as suggested by the 2-hit theory of cancerogenesis.^{34, 35} Nevertheless, it has been suggested that IDH mutations are an early event in a variety of myeloidneoplasias like myelodisplastic syndrome and myeloproliferative neoplasms (MPN).^{35, 36} In patients with MPN, the acquisition of IDH mutations predicts an increased risk of progression to secondary AML, potentially serving as a marker for early stage transformation.^37–39 Also, the fact that IDH mutations are stable during the course of the disease supports the presumption that their emergence is an early event in malignant transformation.

Even though the prognostic significance of IDH mutations has been extensively studied, there are only few reports about their value in MRD monitoring. Thus, Gross et al.⁴⁰ and Jeziskova et al.²³ each presented four patients with IDH1 and IDH2 mutations, followed by the investigations of Chou et al.^{15, 21} In all three studies, as in our nine IDH⁺ patients who were available for sequential analysis, the mutation was lost during CR and reappeared at relapse of the disease as the same type of mutation. Moreover, none of the patients acquired new IDH mutations during relapse.^{15, 21, 23, 40} In our study, we registered two IDH⁺ patients retaining the mutation in CR and during the whole follow-up. Chou et al.²² explained a similar finding through the hypothesis that IDH mutations are important in maintaining the leukaemia phenotype through cooperation with other oncogenic mutations, but alone are not sufficient for leukaemogenesis in vivo.

IDH1 and IDH2 mutations have significant potential as MRD markers, assuming that the method applied meets the sensitivity criteria for MRD detection. The usual method for discovering IDH mutations is PCR-followed by direct sequencing, with a sensitivity of about 20%.^{8, 14, 15, 18} Based on this and the fact that IDH mutations are heterozygous, the application of more sensitive methods, such as real-time PCR specific for a given mutation, should be considered for monitoring therapy response and early relapse.

In conclusion, acquired IDH mutations are common abnormalities in AML-NK. They confer an adverse effect, especially in patients lacking NPM1 mutations. In combination with other molecular markers, IDH mutational status can lead to an improved risk stratification approach for AML-NK patients. Moreover, IDH mutations are stable during the course of the disease and can be potentially used as markers for MRD detection. This could be especially important if specific treatment with IDH inhibitors is introduced in everyday practice.