INTRODUCTION
Acute myeloid leukemia (AML) is a heterogeneous group of neoplastic diseases with a large variability in regard to the clinical course and response to treatment, as well as in their genetic and molecular basis (more than 300 chromosomal translocations and genetic mutations have been described). More than one mutagenic event is probably necessary to give origin to the disease, encompassing cell proliferation mechanisms (class I mutations, such as BCR-ABL, FLT3, RAS, c-Kit, PTPN11, NF1, TEL-PDGRß ) and differentiation block (class II mutations, such as CBFßMYH11, AML1-ETO, TEL-AML1, PML-RARA, MLL, NUP98-HOXA9, PU.1, C/CEP α, AML1, AML-AMP19, CEBPA, NPM1 )^{(1,2)}.
In addition to age, cytogenetic and molecular changes present upon diagnosis are the main variables associated to AML prognosis. Until five years ago, cytogenetic abnormalities defined three risk groups for AML patients younger than 60 years. Approximately 25% of patients belonged to the low risk group, with t(15;17), t(8;21) and inv(16); 25 to 30% of the unfavorable risk, with the following gene rearrangement: MLL, t(6;9), t(9;22), monosomy/ deletions of chromosomes 5 and 7, inv(3)(q21q26) and complex karyotypes. Finally, 50 to 60% of patients had intermediate risk with t(9;11), +8, -Y and normal karyotype, the latter accounting for up to 50% of the cases^{(3–5)}. The estimated five-year survival for those with cytogenetic subtypes for low, intermediate and high risk was 55, 38 and 11%, respectively, in a series of 609 AML patients aged under 60 years^{(4)}.
Elderly patients traditionally have a higher percentage of cytogenetic abnormalities related to bad prognosis (up to 51%) and a lower percentage of good prognosis (about 4%)^{(6)}. There is a drop in the survival curves of those aged over 60 years for the same cytogenetic subtype, with the exception of acute promyelocytic leukemia (APL), according to a study of 1225 AML patients ^{(5)}. Farag et al. also demonstrated that for the population older than 60 and treated with classic chemotherapeutic regimens, only 6% were alive after 5 years^{(7,8)}.
In the last few years, many genetic abnormalities were discovered in normal karyotype AML, especially mutations in the NPM1 (nucleophosmin), FLT3 (fmsrelated tyrosine kinase 3); CEPBA (CCAAT/enhancer binding protein α); MLL PTD (myeloid-lymphoid or mixed-lineage leukemia), NRAS -(neuroblastoma RAS viral oncogen), BAALC (brain and acute leukemia gene), ERG (v-ets erythroblastosis virus E26 oncogene-like) genes, among others^{(9)}. Approximately 45% of AML cases have normal karyotype; in that, the mutations of NPM1 and FLT3 genes are the most prevalent, corresponding to 45 to 55% and 35 to 45% of the cases, respectively^{(10)}.
FLT3 gene, in chromosome 13q12, encodes a receptor with tyrosine-kinase activity related to the activation of the cell proliferation signaling pathways, which is intensely expressed in the initial phases of myeloid precursor cells. Mutations like FLT3-ITD consist of in-tandem insertions of variable length in the region that encodes the receptor juxtamembrane domain. On the other hand, the FLT3-TKD mutation is punctual and affects the tyrosine-kinase domain. Both result in constant tyrosine-kinase activity. FLT3-ITD mutation appears in 35 to 45% of AML cases with normal karyotype, while the FLT3-TKD mutation is found in less than 5%^{(10)}.
The NPM1 gene, in chromosome 5q35, encodes a nucleolar phosphoprotein that makes the transportation between nucleus and cytoplasm, and is directly involved in the regulation and stability of nuclear proteins. The most frequent mutation is the duplication of four pairs of bases in exon 12 (85% of cases), but other types of insertion of four pairs of bases can also occur in the same region. This mutation causes the aberrant location of protein NPM1 in cytoplasm^{(11)}.
It has been demonstrated that normal karyotype AML with mutation in the NPM1 and CEBPA genes or in both have favorable prognosis, whilst mutations in gene FLT3 bear unfavorable prognosis. The cases with simultaneous mutations in genes FLT3 and NPM1 correlate to intermediate prognosis^{(12)}.
Based on such knowledge, the World Health Organization (WHO), in 2008, classified AML into different groups, including AML with recurrent genetic abnormalities that comprise nine subtypes, two of them being temporary entities (Chart 1)^{(13,14)}.
AMl with recurrent genetic abnormalities | |
AMl with t(8;21)(q22;q22)- RUNX1T1-RUNX1 | |
AMl with inv(16)(p13.1q22) or t(16;16)(p13.1;q22)- CBFß-MYH11 | |
Acute promyelocitic leukemia with t(15;17)(q22;q21)- PML-RARA | |
AMl with t(9;11)(p22;q23)- MLLT3-MLL | |
AMl with t(6;9)(p23;q34)- DEK-NUP214 | |
AMl with inv(3)(q21;q26.2) or t(3;3)(q21;q26.3) RPN1-EVI1 | |
AMl (megakaryoblastic) with t(1;22)(p13;q13)- RBM15-MKL1 | |
AMl with NPM1 mutation – temporary entity | |
AMl with CEBPA mutation - temporary entity | |
AMl with alterations related to myelodysplasia | |
Myeloid neoplasms related to therapy | |
Non specified AMl | |
Myeloid sarcoma | |
Myeloid proliferations related to Down syndrome | |
Blastic plasmacytoid dendritic cell neoplasm |
AMl: acute myeloid leukemia.
Therefore, new groups of genetic risk have been recently defined for AML, as depicted in chart 2.
Favorable | t(15;17)(q22;q21)-PML-RARA |
t(8;21)(q22;q22)-RUNX1T1-RUNX1 | |
inv(16)(p13.1q22) or t(16;16)(p13.1;q22)-CBFß-MYH11 | |
Mutated NPM1 and with no FLT3-ITD (normal karyotype) | |
Mutated CEBPA (normal karyotype) | |
Intermediate I | Mutated NPM1 and with FLT3-ITD (normal karyotype) |
Wild-type NPM1 and with FLT3-ITD (normal karyotype) | |
Wild-type NPM1 and with no FLT3-ITD (normal karyotype) | |
Intermediate II | t(9;11)(p22;q23)-MLLT3-MLL |
Unfavorable or favorable cytogenetic abnormalities | |
Unfavorable | inv(3)(q21;q26.2) or t(3;3)(q21;q26.3) -RPN1-EVI1 |
t(6;9)(p23;q34)-DEK-NUP214 | |
t(v;11)(v;q23)-MLL rearrangement | |
-5/5q-, –7/7q | |
Complex karyotype |
Other genetic abnormalities not listed in chart 2 also seem to predict survival. Mutations in c-Kit gene seem to be associated to a worse prognosis in AML with t(8;21) or inv(16), also called CBF-AML (core binding factor)^{(2,15)}. Monosomic karyotype, defined as the karyotype with two or more autosomal monosomies or one autosomal monosomy associated to the structural anomaly, has also been associated to the worst risk group^{(16,17)}.
Multiparametric flow cytometry is essential to characterize myeloid neoplasias and analyze a large number of cells in a short period of time, characterizing many antigens per cell. The identification of leukocyte differentiation antigens in the membrane and cytoplasm, allows for the detection of mixed, aberrant phenotypes, and the follow-up of minimal residual disease. The expression of certain antigens, such as CD7, CD11b, CD14, CD56 and CD34 may be associated to adverse prognosis. Aberrant phenotypes are found in at least 75% of AML^{(13)}. Immunophenotyping demonstrated peculiar features for AML with mutated NPM1, that is, antigen expression of CD13, CD33 and MPO concurrent with the expression of monocytic antigenes CD14 and CD11b, and the absence of the expression of CD34^{(13)}.
OBJECTIVE
To study the rate of mutations that may be associated to a good or bad prognosis, as well as their relation to karyotype and immunophenotype studies in patients with AML.
METHODS
The Special Technique Laboratory of the Hospital Israelita Albert Einstein (HIAE) receives samples of AML patients from different treatment centers for immunophenotyping, cytogenetic and molecular studies. As from 2009, after signing the informed consent, 30 bone marrow samples from newly diagnosed or relapsed AML patients were submitted to study of FLT3-ITD, FLT3-TKD and NPM1 mutations. All samples were submitted to immunophenotypic study and 25 of them to kariotyping.
The immunophenotyping study was performed using cells labeled with monoclonal antibodies for proliferative panel (CD2, CD4, CD7, CD10, CD11b, CD11c, CD13, CD14, CD15, CD19, CD20, CD22, CD33, CD38, CD34, CD56, CD64, CD71, CD117, HLA-DR, glycophorin A, CD3 c, MPOc, TdT and CD79a). After preparing with ammonium chloride (hemolytic buffer), flow cytometry was performed (Epics XL-MCL or FC-500 – Beckman Coulter).
Cytogenetic study was carried out in 24- and 48-hour cultures, with no stimulating agents, submitted to G-banding and described according to the international nomenclature (ISCN 2009).
For analysis of the mutations in FLT3 and NPM1 genes, the DNA of the samples was extracted using the QIAmp DNA Blood Mini Kit (Quiagen), purified (EXOSAP) and submitted to PCR for amplification, using specific primers and labeled for the FLT3-ITD (exon 14/15) and NPM1 mutations. Capillary electrophoresis was utilized for analyses of FLT3-ITD and NPM1 per fragment size. To investigate FLT3-TKD mutations, a new PCR reaction was performed with specific primers for the Nested-PCR, followed by sequencing. The analysis of the FLT3-ITD and NPM1 mutations were performed by means of the software GeneMapper, and the sequencing, by SeqScape.
RESULTS
The description of the immunophenotypical, molecular, and karyotypicl findings is depicted on chart 3.
NUMBER | SEX | AGE | NPM1 | FLT3 | KARIOTYPE | Prognostic genetic Classification | Lineage (CD) | Maturation (CD) | Immaturity | Activation | Proliferation | Lineage B (CD) | Lineage T/NK (CD) | |||||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
ITD | 13 | 33 | 117 | MPO | 11b | 11c | 15 | 14 | 15/64 | 4 | 34 | TdT | 38 | HLA-DR | 71 | 19 | 20 | 22 | 79a | 2 | 7 | 56 | ||||||
1 | FEM | 65 | POS | POS | 46,XX[20] | intermedate-1 | POS | POS | POS | NEG | POS | NEG | POS | NEG | - | POS | NEG | NEG | POS | POS | NEG | NEG | - | - | NEG | POS | POS | NEG |
2 | FEM | 34 | POS | POS | 46,XX[20] | intermediate-1 | POS | POS | NEG | - | POS | POS | POS | NEG | - | - | NEG | - | POS | NEG | POS | - | - | - | - | NEG | NEG | NEG |
3 | FEM | 30 | POS | POS | 46,XX[20] | intermediate-1 | POS | POS | POS | POS | NEG | POS | POS | NEG | - | POS | NEG | - | POS | POS | POS | NEG | - | - | NEG | NEG | NEG | NEG |
4 | MALE | 30 | POS | NEG | 46,XY[20] | favorable | POS | POS | POS | POS | NEG | POS | POS | NEG | - | POS | NEG | - | POS | NEG | POS | NEG | - | - | NEG | NEG | NEG | NEG |
5 | FEM | 4B | POS | NEG | 46,XY[20] | favorable | POS | POS | POS | POS | NEG | POS | POS | NEG | - | NEG | POS | - | POS | POS | NEG | NEG | - | - | NEG | NEG | POS | NEG |
6 | FEM | 72 | NEG | POS | 46,XX[20] | intermediate-1 | POS | POS | POS | POS | - | POS | POS | NEG | - | NEG | POS | - | POS | POS | POS | NEG | - | - | NEG | NEG | NEG | NEG |
7 | FEM | 57 | NEG | NEG | 46,XX[20] | intermediate-1 | POS | POS | POS | POS | NEG | POS | POS | NEG | - | NEG | POS | - | POS | POS | POS | - | - | - | NEG | NEG | NEG | NEG |
8 | FEM | 32 | NEG | NEG | 46,XX[20] | intermediate-1 | POS | POS | POS | POS | POS | POS | POS | NEG | - | POS | NEG | - | POS | POS | POS | NEG | - | - | NEG | NEG | NEG | NEG |
9 | MALE | 73 | NEG | NEG | 46,XY[20] | intermediate-1 | POS | POS | POS | POS | POS | NEG | POS | NEG | - | POS | NEG | - | POS | POS | POS | NEG | - | - | NEG | NEG | POS | NEG |
10 | FEM | 44 | NEG | NEG | 46,XX, inv (9) (p12q13)[20] | intermediate-1 (normal varient) | POS | POS | POS | POS | NEG | NEG | NEG | NEG | - | NEG | POS | NEG | POS | NEG | POS | NEG | - | - | NEG | NEG | NEG | NEG |
11 | FEM | 30 | NEG | POS | 46,XX, t(15;17)(q22;q21) [14] | favorable (LPA) | POS | POS | POS | POS | NEG | NEG | POS | NEG | - | NEG | POS | NEG | POS | NEG | POS | NEG | - | - | NEG | POS | NEG | NEG |
12 | FEM | 11 | NEG | POS | 46,XX,t(15;17)(q22;q21)[16]/46,XX[4] | favorable (LPA) | POS | POS | POS | POS | NEG | NEG | NEG | NEG | - | NEG | NEG | - | FOG- | NEG | POS | POS | NEG | NEG | NEG | NEG | NEG | NEG |
13 | FEM | 30 | NEG | NEG | 46,XX,t(15;17)(q22;q21)[7],46,XX[13] | favorable (LPA) | POS | POS | POS | POS | NEG | NEG | NEG | NEG | - | NEG | NEG | - | POS | NEG | NEG | NEG | - | - | NEG | NEG | NEG | NEG |
14 | FEM | 27 | NEG | NEG | 46,XX,t(15;17)(q22;q21) [20] | favorable (LPA) | POS | POS | POS | POS | NEG | NEG | NEG | NEG | - | NEG | NEG | - | POS | NEG | NEG | NEG | - | - | NEG | NEG | NEG | NEG |
15 | FEM | 66 | NEG | NEG | 46,X,del(X)(q24),t(4;21;8)(q21;q22;q22)[21] | favorable (varient (8;21)) | NEG | POS | POS | NEG | NEG | POS | POS | NEG | - | POS | NEG | - | POS | POS | NEG | NEG | - | - | NEG | NEG | NEG | POS |
16 | MALE | 11 | NEG | NEG | 46,XY, del(16)(q22)[20] | intermediate-2 | POS | POS | POS | POS | NEG | NEG | NEG | NEG | - | NEG | POS | - | POS | POS- | POS | NEG | - | - | NEG | NEG | NEG | NEG |
17 | FEM | B | NEG | NEG | 46,XX, add(2)(q37)[20] | inetrmediate-2 | POS | POS | POS | POS | NEG | NEG | NEG | NEG | - | NEG | POS | - | POS | POS | NEG | NEG | - | - | NEG | NEG | NEG | POS |
18 | MALE | 59 | NEG | NEG | 46,XY,del(3)(p21)[18] | inetrmadiate-2 | NEG | POS | NEG | - | NEG | NEG | NEG | NEG | - | NEG | NEG | - | POS | NEG | POS | NEG | - | - | - | POS | POS | NEG |
19 | FEM | 48 | POS | POS | 47,XX,+5[3]/46,XX[17] | intermadiate-2 | POS | POS | POS | POS | NEG | NEG | NEG | NEG | - | POS | POS | - | POS | POS | POS | NEG | - | - | NEG | NEG | NEG | NEG |
20 | FEM | 68 | NEG | NEG | 46,XX,t(6;19)(q 16;p 12) [5]/46,X[15] | intermadiate-2 | POS | POS | POS | NEG | NEG | POS | NEG | NEG | - | NEG | NEG | - | POS | POS | POS | NEG | - | - | NEG | NEG | POS | NEG |
21 | MALE | 61 | NEG | NEG | 46,XY,t(3;3)(q21;q26)[19]/46,XY[1] | unfavorable | POS | POS | POS | NEG | POS | NEG | NEG | NEG | - | NEG | POS | POS | POS | POS | POS | NEG | - | - | NEG | NEG | POS | NEG |
22 | MALE | 15 | NEG | NEG | 46,XY,t(6;9)(p23;q34)[20] 46,XY, inv(9) | unfavorable | POS | POS | NEG | - | POS | POS | POS | NEG | - | POS | NEG | - | POS | POS | NEG | NEG | - | - | - | NEG | NEG | NEG |
23 | MALE | 9 | NEG | NEG | p21q13), t(11;?)(q23;?) [19] | unfavorable | POS | POS | POS | NEG | NEG | NEG | NEG | NEG | - | NEG | POS | - | POS | POS | POS | NEG | - | - | NEG | NEG | NEG | NEG |
24 | FEM | 61 | NEG | NEG | 46,XX,del(7)(q21)[7]/46,XX[13] | unfavorable | POS | POS | POS | POS | NEG | NEG | NEG | NEG | - | POS | POS | - | POS | NEG | NEG | NEG | - | - | NEG | NEG | NEG | POS |
25 | MALE | 20 | NEG | NEG | 46,XY,t(9;18)(q34;q22),t(13;20)(q12;q13.2)[15]/46,XY[5] | unfavorable | POS | POS | POS | - | - | POS | POS | NEG | - | POS | POS | - | POS | POS | POS | NEG | - | - | - | NEG | NEG | POS |
26 | MALE | 61 | POS | POS | - | POS | POS | NEG | POS | NEG | NEG | NEG | NEG | - | NEG | NEG | - | NEG | NEG | NEG | NEG | - | - | NEG | NEG | NEG | POS | |
27 | FEM | 89 | POS | POS | - | POS | POS | POS | NEG | NEG | NEG | NEG | NEG | - | NEG | POS | - | POS | POS | POS | NEG | - | - | NEG | NEG | POS | NEG | |
28 | FEM | 51 | POS | NEG | - | POS | POS | POS | POS | POS | POS | POS | NEG | - | POS | NEG | - | POS | POS | POS | NEG | - | - | NEG | NEG | POS | NEG | |
29 | FEM | 94 | POS | NEG | - | POS | POS | POS | NEG | NEG | NEG | NEG | NEG | - | NEG | POS | - | POS | POS | POS | NEG | - | - | NEG | NEG | POS | NEG | |
30 | FEM | 61 | NEG | POS | - | POS | POS | POS | POS | - | NEG | POS | NEG | - | NEG | POS | - | POS | POS | POS | NEG | - | - | NEG | NEG | POS | NEG |
In the cases studied, NPM1 gene mutation was found in 10 out of 30 samples (33.3%), the same been observed for the FLT3-ITD mutation. The coexistence of both occurred in six cases. No sample was positive for the FLT3-TKD mutation.
Of the 25 samples with karyotypes analyzed, 10 had normal karyotypes (40%). When only normal karyotypes were analyzed, 50% presented the mutation in NPM1 gene and 40% FLT3-ITD mutation. The concurrent presence of both was diagnosed in 30% of the cases.
Among the 15 samples diagnosed with abnormal karyotype, 4 presented karyotypes with t(15;17) and one with the variant t(8;21), considered of good prognosis. Five samples presented karyotypes of unfavorable prognosis, t(3;3), t(6;9), del(7)(q21), 11q23 rearrangement and one complex. Five other cases considered of intermediate prognosis such as del(16)(q22), add(2)(q37), del(3)(p21), 5 trisomy and t(6;19) were also found.
Three cases of abnormal karyotypes also presented molecular alterations, two with t(15;17) and FLT3-ITD mutation, and one 5 trisomy with mutated FLT3-ITD and NPM1.
According to our study, of the 25 cases with karyotype study, 28% were classified as favorable prognosis, among which 8% had normal karyotype and mutated NPM1, and 20% had abnormal karyotype (16% t(15;17), and 4% t(8;21). Thirty-two percent were classified as intermediate-1, all with normal karyotype and 12% mutated NPM1 and FLT3-ITD, 4% FLT3-ITD and 16% wild for both mutations. The remaining cases, all with abnormal karyotype, were stratified as intermediate-2 (20%) and unfavorable (20%) (Figure 1).
The immunophenotype study demonstrated that 18 cases (60%) expressed non-myeloid lineage-associated antigens: CD7 in 10 cases, CD56 in 5 cases and CD11b in 7 cases; in that, four cases having concurrence of CD7 and CD11b. The expression of CD56 was detected in 16.6% of AML cases and 20% of cases with FLT3 mutation.
The immunophenotype study of four cases with wildtype NPM1 and FLT3 mutation (2 with normal karyotype and 2 with no karyotype study) demonstrated, in two of them, absence of CD34 expression. Of the 6 cases with mutated NPM1 and FLT3-ITD genotype (3 with normal karyotype, 1 abnormal, 2 with no karyotype study), four had no expression of CD34. Only one of 4 cases of wildtype FLT3-ITD and NPM1 (1 with normal karyotype, 2 with t(15;17) and 1 with no karyotype study) had absent CD34 expression.
DISCUSSION
Although a small number of samples from AML patients was studied, the rates observed for FLT3 and NPM1 mutations were similar to those found in the literature, for all subtypes of AML, as well as for AML with normal karyotypes.
In a publication with a national series, a total of 43.7% in NPM1 mutations were observed in AML subtype with normal karyotype, a rate similar to the one observed in the present study ^{(18)}. The incidence of 33.3% FLT3-ITD mutation was slightly higher that reported by LucenaAraujo (23.6%), being found in the many subtypes of AML, including APL, these data being consonant to the international and national literature^{(18,19)}.
Mutation type TKD was not detected, corroborating the less prevalent finding of this mutation in Brazilian series when compared to American or European ones^{(18–20)}.
In regard to immunophenotype, the small series hindered comparison with data found in the literature, such as smaller expression of CD56 in the subtype with mutated FLT3 and absence of CD34 expression and HLA-DR in the AML with mutated NPM1. In this study, the rate of expression of CD56 was similar to the one observed in AML with FLT3 mutation and also one of the patients with NPM1+/FLT3- AML presented blast cells with the expression of CD34 and HLA-DR.
Considering the above, it may be inferred that the diagnostic routine should encompass new genetic markers for correct prognostic stratification and treatment option in AML.