Genetic Pathways in the Pathogenesis of Therapy-Related Myelodysplasia and Acute Myeloid Leukemia

A pronounced heterogeneity of myelodysplasia (MDS) and acute myeloid leukemia (AML) was first demonstrated by observation of their many alternative recurrent chromosome aberrations.^1,–5 These changes have, in many cases, been shown directly to result in gene rearrangements participating in leukemogenesis.^6,7 Cytogenetics have become of crucial importance for diagnosing and treating patients with MDS and AML. Based on the presence of specific chromosome aberrations, different genetic pathways have been proposed in t-MDS and t-AML.^8,9 During recent years, point mutations or internal tandem duplications of many genes important to hematopoiesis have been demonstrated in MDS and AML. Their association to the recurrent chromosome aberrations and distribution in the different genetic pathways, as well as their mutual cooperation, will be discussed in this review.

Two groups of patients with MDS and AML must be considered: patients with de novo disease and patients with therapy-related disease. Whereas most cases of de novo disease present without any knowledge of prior leukemogenic exposures, most cases of t-MDS and t-AML are observed in patients treated with highly mutagenic cytostatic drugs or irradiation for various types of primary tumor. At present, 80% to 90% of patients with MDS or AML diagnosed at major centers are de novo cases, whereas 10% to 20% represent therapy-related cases.

Three main cytogenetic subgroups of MDS and AML are observed (Table 1 ).^1,–5 The first subgroup includes patients with recurrent unbalanced aberrations and either loss or gain of chromosome material, but without any of the recurrent balanced aberrations. Loss of various parts of the long arm or loss of whole chromosomes 5 or 7 (5q−/−5, 7q−/−7) are frequently observed, as are gain of a whole chromosome 8 (+8). The second cytogenetic subgroup comprises patients with one of the recurrent balanced aberrations with chromosome rearrangement without any visible loss or gain of chromosome material. The most common balanced aberrations are the reciprocal translocations involving chromosome bands 11q23, 21q22, 17q21 or inv(16)(p13q22). The third cytogenetic subgroup contains patients with a normal karyotype.

The distribution of patients between the three cytogenetic subgroups differs in therapy-related^8,10,11 and de novo MDS and AML^1,–5 (Table 1 ). In de novo MDS, more than 50% present a normal karyotype and 15% to 25% present unbalanced aberrations, whereas balanced translocations are rare. In de novo AML, up to 50% present a normal karyotype, 15% to 25% present an unbalanced aberration, often 5q−/−5, 7q−/−7, or +8, and 15% to 20% present a balanced translocation.

In t-MDS, unbalanced aberrations, particularly 5q−/−5 or 7q−/−7, are observed in 50% to 70% of the patients. A normal karyotype is observed in just 5% to 10% of the cases, and balanced aberrations are rare. In overt t-AML, unbalanced aberrations with 5q−/−5 and 7q−/−7 are also common, 10% to 15% present a normal karyotype, and 15% to 20% present a balanced translocation or inversion.

Therapy-related MDS and t-AML are of particular importance to study for several reasons:

1. They represent the most serious long-term complications to current cancer therapy.

2. They are directly induced by chemically well-defined agents or irradiation with well-known cellular effects.

3. They present the same chromosome aberrations and gene mutations as de novo MDS and AML, allowing extrapolation of results from one to the other type of disease.

4. An early stage of MDS with refractory cytopenia is often diagnosed in therapy-related disease, because most patients are followed thoroughly after intensive chemotherapy or irradiation. In de novo AML, such information is often lacking.

Interestingly, in t-MDS or t-AML, the abnormalities 5q−/−5 and 7q−/−7 are closely related to previous therapy with alkylating agents.^10,–12 The karyotypes are frequently complex with many unidentified marker chromosomes. Balanced translocations, on the other hand, are mainly observed in t-AML following therapy with topoisomerase II inhibitors, and are often present as the sole abnormality.¹² Finally, patients with t-AML and a normal karyotype have often received radiotherapy only or treatment with antimetabolites or other cytostatic drugs without any well-documented leukemogenic potential.

The recurrent balanced chromosome aberrations often result in chimeric rearrangements between genes encoding hematopoietic transcription factors and various partner genes.^6,7 The most important rearranged transcription factors genes include the MLL at 11q23, the AML1 at 21q22, the RARA at 17q21 and the CBFB at 16q22. These gene rearrangements lead to a dominant loss-of-function of the transcription factor, to impairment of differentiation, and to development of leukemia-like disease in transgenic mice.¹² This type of abnormality has been designated Class II mutations in leukemogenesis, and mutations of the NPM1 gene possibly also belong to this class.

Until recently, the critical genetic consequences of unbalanced chromosome aberrations in MDS and AML have remained unknown. Initially, deletions of chromosome band 17p13 or loss of a whole chromosome 17 harboring the p53 gene were shown to be associated with point mutation of p53.^13,–15 Recently, 5q−/−5 was related to haploinsufficience of the EGR1 gene in one study,¹⁶ and in another study to haploinsufficience plus promoter methylation of the gene encoding α-catenin (CTNNA1).¹⁷ Both genes are located on chromosome band 5q31. In another important study of MDS cells with −7, a constitutive activation of the STAT1/STAT5 signal transduction pathway was observed following stimulation with granulocyte colony-stimulating factor (G-CSF).¹⁸ This effect was attributed to an increased proportion of the G-CSF receptor isoform 4, which fails to internalize following G-CSF stimulation. The relationship between the shift in isoform of the receptor and the loss of a chromosome 7 remains unclear. These new studies may provide further insight into the complicated mechanisms of leukemic transformation and offer new therapeutic possibilities.

Point mutations of the tumor suppressor gene p53 have previously been studied extensively in solid tumors and have also been observed in de novo MDS and AML.^13,15 In t-MDS and t-AML, they represent the most frequent single genetic abnormality, detected in 20% to 30% of the patients^14,19,20 (Table 2 ). The p53 mutations, often with loss of heterozygosity of the gene, are closely associated with the cytogenetic defects 5q−/−5¹⁹ and 17p−/−17.^13,14,19 Patients with p53 mutations characteristically present complex karyotypes and complicated chromosome rearrangements^14,19 with duplication or amplification of chromosome bands 11q23 and 21q22 encompassing the MLL and the AML1 genes,²¹ and “sandwich-like” marker chromosomes made of material from at least three different chromosomes.²² These patients have an extremely poor prognosis.

Besides mutations of transcription factors (Class II mutations) and mutations of p53, Class I mutations are common in MDS and AML. These mutations activate tyrosine kinases, such as FLT3, cKIT, cFMS or JAK2, or genes further downstream in the RAS-BRAF-MEK-ERK signal transduction pathway, such as NRAS, KRAS, BRAF or PTPN11 (Table 2 ). These mutations result in constitutive activation of cell cycling and proliferation, and they have been designated as Class I mutations. They are often considered as late events in leukemogenesis and possibly cooperate with Class II mutations in leukemogenesis,^23,24 as discussed below. Mutations of RAS are common in monocytic subtypes of AML²⁵ and in chronic myelomonocytic leukemia (CMML)²⁶ and even more frequent in juvenile myelomonocytic leukemia (JMML),²⁷ in which mutations of the PTPN11 gene are also common.²⁸ Interestingly, RAS was recently shown to favor monocytic lineage selection of hematopoietic progenitors.²⁹ 

The issue of epigenetic changes in MDS and AML is becoming increasingly important. As discussed above, promoter methylation of the CTNNA1 gene may be an important abnormality underlying 5q–. Another example is promoter methylation of the p15INK4B gene. This was observed in 55 of 81 patients with t-MDS or t-AML (Figure 1; see Color Figures, page TK),³⁰ significantly associated with 7q−/−7, and the frequency increased with progression from refractory anemia (RA) to RA with excess blasts in transformation (RAEBT).

Eight alternative genetic pathways have been proposed in t-MDS and t-AML (Figure 1; see Color Figures, page 510):^8,9 

Pathway I comprises patients with 7q−/−7 but normal chromosomes 5 and without balanced aberrations. Such cases characteristically present as t-MDS and are observed following therapy with alkylating agents. They frequently have point mutations of AML1,^31,–33 which are significantly associated with subsequent progression to overt t-AML.

Pathway II includes patients with 5q−/−5, but without balanced aberrations. They present as t-MDS or t-AML. As discussed above, most of these cases show mutation of p53 and complex chromosome rearrangements. They are primarily observed after therapy with alkylating agents. Occasionally, these patients also have the cytogenetic defects 7q−/−7.

Pathway III is represented by patients with overt t-AML and balanced translocations involving the chromosomal band 11q23, resulting in chimeric rearrangements between the MLL gene and one of its numerous alternative partner genes. These overt leukemias are often of FAB subtypes M4 or M5 and significantly associated with previous therapy with topoisomerase II inhibitors, primarily epipodophyllotoxins. In such patients, mutations of NRAS, KRAS or BRAF are common.^25,34 

Pathway IV includes patients with balanced translocations to chromosome band 21q22 or inv(16), leading to chimeric rearrangement between the core binding factor genes AML1 or CBFB. Except for cases with the t(3;21), such patients characteristically present as overt t-AML and frequently follow therapy with anthracyclines. Patients of this type with de novo AML show point mutation of the receptor tyrosine kinase cKIT as the most common additional mutation.^35,36 

Pathway V comprises patients with acute promyelocytic leukemia and chimeric rearrangement of the RARA gene at 17q21. Due to their responsiveness to retinoic acid and favorable prognosis, they are grouped separately. In t-AML, such cases have often been observed related to previous therapy with mitoxantrone for breast cancer, and, like patients with de novo acute promyelocytic leukemia, they often present internal tandem duplications of FLT3.

Pathway VI is represented by patients with t-MDS or t-AML and chimeric rearrangement of the NUP98 gene on 11p15. Such cases are in most instances related to therapy with topoisomerase II inhibitors, but so far no other specific mutations have been observed to cluster in patients belonging to this pathway.

Pathway VII includes patients with a normal karyotype. Previously, nothing was known about their molecular biology. Recently, however, point mutations of the NPM1 gene,^37,–39 internal tandem duplications of FLT3,^15,40,41 and point mutations of CEBPA^42,–44 have been observed, particularly in patients with de novo AML and a normal karyotype. These abnormalities all contribute differently to the prognosis of the disease. Also, point mutations of RAS³⁴ and internal tandem duplications of MLL are occasionally observed in patients with t-AML and a normal karyotype. In t-MDS and t-AML, a normal karyotype is often observed in clinically atypical cases without association to any specific type of previous therapy.

Pathway VIII in t-MDS and t-AML comprises patients with other, often unique chromosome aberrations. Patients with trisomy 8 may belong to this pathway or represent a separate entity. Patients in pathway VIII with “other aberrations” are not associated with any specific type of previous therapy and, for unknown reasons, only rarely present mutations of genes in the RAS-BRAF signal transduction pathway or mutations of transcription factors, at least in our series of patients.

Whereas mutations of genes in the RAS-BRAF signal transduction pathway (Class I mutations), as well as mutations of genes encoding transcription factors and the transcription regulatory gene NPM1 (Class II) are negatively associated within each class, an association, possibly indicating cooperation, is often observed between Class I and Class II mutations.^23,24 This was recently supported by our investigation of 140 patients with t-MDS or t-AML.⁹ In this study, 35 patients presented a Class I mutation, and a Class II mutation was observed in 59 patients, whereas 25 patients presented a simultaneous mutation of both classes (P = .0001) (Mutations of the NPM1 gene in therapy-related myelodysplasia and acute myeloid leukemia, manuscript submitted August 2007). Many of the associations observed in t-AML, such as the NPM1-FLT3, AML1-cKIT and RARA-FLT3 combinations, have previously been emphasized in de novo AML.

De novo and therapy-related MDS and AML are heterogeneous diseases with identical cytogenetic abnormalities and gene mutations, although some of these are observed with different frequencies in the two subsets of patients. This suggests that therapy-related and de novo cases in fact are identical disorders, allowing to extrapolate results from one to the other type of disease. Therapy-related MDS has a high potential for transformation to overt t-AML, which is associated with point mutations of the AML1 and the RAS genes.

Three cytogenetic subtypes are observed. Patients with unbalanced aberrations, primarily 5q−/−5 or 7q−/−7, represent the first subtype. They often present as MDS and disclose a complex karyotype with point mutations of p53 or AML1. In the therapy-related subset and perhaps also in de novo disease, they are associated with exogenous respectively endogenous alkylating exposures. Patients with the recurrent balanced translocations or inversions represent the second subtype. Such cases often present as overt AML and arise, at least in the therapy-related subset, as illegitimate gene recombinations related to the activity of topoisomerase II. Finally, patients with a normal karyotype comprise the third subtype. In t-MDS and t-AML, such cases are occasionally observed after radiotherapy only, but often their etiology is unknown.

Three general types of gene mutation are observed in MDS and AML: Class I, activating mutations of genes in the receptor tyrosine kinase RAS-BRAF pathway, leading to increased cell proliferation; Class II, inactivating mutations of genes encoding transcription factors leading to disturbed cell differentiation; inactivating mutations of the p53 tumor suppressor gene possibly represent a third class of mutations.

At least eight different genetic pathways can be outlined in t-MDS and t-AML, defined by specific chromosome aberrations. The various other genetic changes cluster differently in these pathways. Some abnormalities are related to presentation of the disease as MDS, others are related to presentation as overt AML. Class I and Class II mutations are associated, suggesting their cooperation in leukemogenesis.