MORPHOLOGY AND CYTOGENETICS IN ACUTE MYELOID LEUKEMIA - FOCUS ON MYELODYSPLASIA RELATED CHANGES

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1 MORPHOLOGY AND CYTOGENETICS IN ACUTE MYELOID LEUKEMIA - FOCUS ON MYELODYSPLASIA RELATED CHANGES Elena-Cristina Selicean 1, Mariana Patiu 1,2, A. Cucuianu 1,2, Delia Dima 1, Minodora Dobreanu 3,4 1 ION CHIRICUTA INSTITUTE OF ONCOLOGY, DEPARTMENT OF HEMATOLOGY, CLUJ-NAPOCA 2 IULIU HATIEGANU UNIVERSITY OF MEDICINE AND PHARMACY, CLUJ- NAPOCA 3 UNIVERSITY OF MEDICINE AND PHARMACY, TARGU-MURES 4 EMERGENCY COUNTY HOSPITAL TARGU MURES Summary Morphology and cytogenetic testing are mandatory investigations in acute myeloid leukemia (AML). The aim of our study was to establish correlations between morphology, cytogenetics, and other biological aspects at diagnosis. We focused our attention on the group with myelodysplasia related changes (MRC), especially due to the fact that classification based on multilineage dysplasia is a disputed matter. Analysis was done on bone marrow samples from patients with acute myeloid leukemia, by microscopy on May Gruenwald Giemsa stained smears and by conventional cytogenetic methods. Statistical analysis was done with the Graph Pad Prism program. Morphological changes specific for t(15;17) and inv(16) were present in our patients. The MRC group differed from AML-NOS (not otherwise specified) by leukocyte count (WBC) and blast percentage, but not by age, hemoglobin or platelet number. The WBC and blast percentage have always been recognized as prognostic factors, even if not always as independent prognostic factors. This means that our observations regarding the correlations of these parameters with MRC cannot be neglected. Even if quantification of dysplasia has a high degree of subjectivism, it seems that classification according o these criteria is still necessary, because this group has some particular characteristics and otherwise, as long as we do not have other clasification means, to many cases would remain in the AML-NOS category. Keywords: acute myeloid leukemia, myelodysplasia related changes, morphology, cytogenetics. cristinaselicean@hotmail.com Introduction The diagnosis of acute myeloid leukemia (AML) was for a long time based only on morphology. The French- American-British group (FAB) classifications in 1976 (Bennett et al, 1976) and 1985 (Bennett et al, 1985) incorporated first only morphologic criteria and then some immunophenotyping data. The major achievements of cytogenetics in this field have been recognized as diagnostic criteria only in the 2001 World Health Organization (WHO) classification (Jaffe et al, 2001) and were further refined in the 2008 revision (Swerdlow et al, 2008). Nowadays we are at the point where cytogenetics, accompanied by molecular biology, are considered the main determinants in AML prognosis and thus the first category in the current classification is assigned to patients with recurrent genetic alterations, two provisional entities defined by normal karyotype and CEBPA and NPM1 mutations being added (Vardiman et al, 2009). If patients with cytogenetic 135

2 alterations do not belong to this group, they may still belong to the the myeloid related changes (MRC)-AML category, where myelodysplastic syndrome (MDS) - like cytogenetics is a criterion, or they may benefit of the assignment to a prognosis group, to aid therapeutic decision making. Remaining cases are still diagnosed according to patient history and morphology. Regarding the current role of morphology, there are some doubts if the morphologic criteria of multilineage dysplasia (MLD) and even if the classification on the basis of FAB criteria in the so called not otherwise specified (NOS) category, are of any value. Our study is an attempt to reclassify a series of AML cases according to the latest WHO criteria and to integrate cytogenetic data with other investigations, in order to analyse subgroups and to identify cases of particular interest. Another aim is to reevaluate morphology according to the WHO 2008 criteria and to compare MRC-AML with multilineage dysplasia (mmrc) to cytogenetic MRC (cmrc)- AML and to AML-NOS. Material and methods We have studied 88 patients admitted to the Department of Hematology of the Oncology Institute "Prof. Dr. Ion Chiricuta" in Cluj between , with a diagnosis of AML. All patients were investigated at diagnosis by complete blood count, blood smear, biochemistry and coagulation studies. Other investigations performed at diagnosis were bone marrow smear, immunophenotyping by flowcytometry, karyotyping and molecular biology (RUNX1/RUNX1T1, MLL-PTD, CBFB/MYH11, FLT3-TKD, PML/RARA, FLT3-ITD, AF9/MLL, NPM1). General characteristics of the group in our study (n = 88) are presented in Table 1. Twenty-one patients were over 65 years old, and 8 patients were in the range of years. Twenty one patients had at presentation leukopenia (<4000/µl), and 44 patients had leukocytosis (>10 000/µl). Of these, 9 patients had a white blood cell count (WBC) over 100,000/µl and 8 patients had WBC in the range of to 100,000 /µl. Most patients (n=77) had anemia at presentation. Hemoglobin level was above 8g/dl in 31 patients and between 6 and 8g/dl in 44 patients. Fifthy-seven patients had a platelet count (Plt) below 50,000/µl, of which 16 under 20,000/µl and 5 below 10,000 /µl. 136 Table 1. General data of the patient group Median Range Age at diagnosis (years) WBC (x 1000/µl) Hemoglobin (g/dl) Platelets (x 1000/µl) Blasts in bone marrow (n=76) % Sex ratio - M/F=1.32 Karyotyping was available in 79 patients, the remaining 9 patients not having enough analyzable metaphases. Seventy-five patients had a bone marrow aspirate at diagnosis. For the remaining cases primary diagnosis was based on the high percentage of blasts in peripheral blood and immunophenotyping. Two of them had a history of MDS and recent bone marrow aspirates on which morphology could be assessed in detail. Morphologic analysis was done on stained bone marrow samples under the microscope at a 200x and 1000x magnification. The morphologic description and blast

3 count was made according to FAB and WHO criteria, reviewed and refined by the the International Working Group on Morphology of MDS (IWGM-MDS) (Mufti et al, 2008), taking into account: cell size, chromatin appearance, cytoplasm appearance, granularity, the presence of Auer rods, positivity and appearance of myeloperoxidase in cytochemistry. We used the May- Grunwald Giemsa stain and myeloperoxidase stain (internal protocol of the Medical Analysis Laboratory of the Hematology Department). In order to quantify dysplastic changes we adapted a method already proposed by Miesmer et al (Miesner et al, 2010), which follows the definitions of Goasguen et al (1992) and the WHO 2008 classification: - Dysgranulopoiesis (DysG): more than 50% of at least 10 polymorphonuclear neutrophils (PMN) agranular or hypogranular, or with hyposegmented nuclei (pseudo Pelger-Huet anomaly), presence of Auer rods. - Dyserythropoiesis (DysE): dysplastic features in at least 50% of 25 erythroid precursors (megaloblastoid aspects, karyorrhexis, nuclear particles, bi or multinuclearity, internuclear bridges) - Dysmegakaryopoiesis (DysM) was diagnosed when 3 megakaryocytes or 50% in at least six cells had dysplastic features (micromegakaryocytes, separated nuclei, very large single nuclei) - Multilineage dysplasia (MLD) was defined by dysplastic features in at least two hematopoietic lineages (Dys2, Dys3) - Trilineage dysplasia (TLD/ Dys3) was diagnosed when DysG, DysE, and DysM in combination were detectable. This approach allowed us to reduce as much as possible the inherent subjectivity of morphological testing. Still we had to face the fact that for some specimens one or two of the hematopoietic lines were not appropriately represented on the available slides to allow quantification. Because of the relative small proportions of our group, we could not afford to exclude these cases and, for statistical reasons, where dysplasia could not be assessed we added these cases to the group without dysplasia (Dys0). Karyotyping was done in the Laboratory of the Medical Clinic III Ulm, Germany, by an internal protocol, on cell cultures of bone marrow samples and Giemsa staining. Description of metaphases was made according to the International System of Cytogenetic Nomenclature (ISCN). Statistical analysis was done by the Graph Pad Prism Program: t test for differences of means and Chi Square/ Fischer s exact test for contingency tables. Differences were considered significant for p<0.05. Results 42 of 79 available karyotypes were normal. 18 cases hade complex karyotypes. Other abnormal karyotypes (n=19) presented numerical abnormalities (+8, -7,- 5, +22, 12+, +X, +14, +13), structural abnormalities: t(8;21), inv(16), t(6;11), t(4;12)(q12;q13), t(15;17), del(9q), del(17) or both structural and numerical abnormalities, as for example t(8;21), -Y; - 17,+mar. Five cases were assigned in the AML class with recurrent genetic abnormalities. (Table 2). 137

4 Table 2. AML with recurrent genetic alterations AML with recurrent genetic alterations AML with t(8:21)(q22;q22); (RUNX1-RUNX1T1) 2/79 AML with inv(16)(p13.1q22) or t (16;16)(p13.1;q22); (CBFB-MYH11) 1/79 Promyelocytic AL with t(15;17)(q22;q12); (PML-RARA) 2/79 AML with t(9;11)(p22;q23); (MLLT3-MLL) 0 AML with t(6;9)(p23;q34); (DEK-NUP214) 0 AML with inv(3)(q21q26.2)or t(3;3)(q21;q26); (RNP1-EVI1) 0 AML (megakarioblastic) with t(1;22)(p13;q13); (RMB15-MKL1) 0 On the basis of karyotyping results, The most often encountered we assigned the patients to prognostic classes, according to the MRC criteria revised in 2010 (Grimwade et al, 2010). Cases with favorable cytogenetic changes were assigned to this group irrespective of dysplastic changes were: - on the granulocytic series: pseudo Pelger Huet abnormality, hypogranularity/partial or total degranulation; nucleo-cytoplasmic maturation asynchronism, Auer rods the presence of other cytogenetic changes, - on the erythroid series: biwhether these were bad or favorable (Table multinuclearity, nuclear budding, 3). internuclear bridges, macro/megaloblastosis Only 5 patients fitted into the - on the megakaryocytic series: favorable prognosis class, but 22 patients in the unfavorable one. Patients who did not belong to either of these groups were considered as intermediate risk. 42 out of these were with normal karyotype and could be further classified according to molecular mutations. Out of the cytogenetic changes micromegakaryocytes, hypolobulation, recognised by the WHO as being myelodysplasia related, we idetified in our group 21 cases: 18 with a complex caryotype, two cases with numerical aberations (1 case -5 ; one case -5/-7) an 1 case with del(9q). Two of the cases with complex karyotype harboured mutations which even isolated would signify MRC: 1 case with -7, del(5) and 1 case with del (5q) del(7q); one case harboured the t(9;22) and two cases had a history of MDS. other lobulation abnormalities The working protocol of our laboratory did not require at the time quantification of dysplasia. Dysplasia was only mentioned and eventually appreciated as: dysplastic elements present/ rare/ frequent, or discrete/ impressive dysplasia on the granulocytic / erythroid / megakaryocytic series. In these circumstances, presence of dysplasia in our group was as follows: - granulocytic series dysplasia (Auer bodies included) - present in 45 patients; not evaluable in 9 patients; absent in the rest - erythroid series dysplasia: present at 30 patients; absent or not evaluable in 27 cases - megakaryocytic series: dysplasia was present in 12 cases, absent or not evaluable in 59 cases. Table 3. Cytogenetic risk groups Prognostic Cytogenetic abnormality Cases (n) Favorable t(15;17)(q22;q21) 2 t(8;21)(q22;q22) 2 inv(16)(p13q22) or t(16;16)(p13;q22) 1 0 Unfavorable 0 abn(3q) [excluding t(3;5)(q21 25;q31 35)] 138

5 inv(3)(q21q26)/t(3;3)(q21;q26) 0 add(5q), del(5q), 5 1(-5) 7, add(7q)/del(7q) 1 (-5, -7) t(6;11)(q27;q23) 1 0 t(10;11)(p11 13;q23) t(11q23) [excluding t(9;11)(p21 22;q23) si 0 Intermediate t(11;19)(q23;p13)] t(9;22)(q34;q11) 0 17 or 1 abn(17p) 0 Complex 18 Cases which were not classified as favorable or adverse: Normal karyotype , x del(9q), del(17) 1 t(4;12) 1 Dysplasia was trilinear in 8 cases, bilinear in 17 cases (13 cases with dysplasia on erythroid and granulocytic series), 1 case with dysplasia on granulocytic and megakaryocytic series and 3 cases with dysplasia on erythroid and megakaryocytic series). 23 cases presented dysplasia only on the granulocytic series and 6 cases only on the erythroid series. 25 cases had no dysplasia described on either series, partially as we already mentioned because of improper aspirates and slides. Reassessment of dysplasia according to the WHO criteria as described in the methods part had as consequence the reassignment of 16 cases from the NOS category to the MRC-AML group. Table 4. Normal and complex karyotypes as encountered within the FAB groups M0 M1 M1/M2 M2 M2/M4 M3 M4 M4/M5b M5 M6 Mx NKT CKT other NKT normal karyotype CKT complex karyotype Thus, out of the 79 cases of the group, 5 were reassigned as AML with recurrent cytogenetic alterations, 21 had MRC (21 cytogenetic and 16 morphologic changes) and 37 cases remained in the NOS category. Normal and complex karyotypes as encountered within the FAB groups are presented in Table 4. The best represented cases in our group, M2 and M4, showed a pattern of cytogenetic abnormalities as depicted in table 5 and Figure

6 Table 5. Cytogenetic findings in the M2 and M4 group NKT CKT +8 +8, ,- t(8;21), -5 t(6;11) t(4;12) t(8;21) 5 -y inv(16) M M Figure 1. Cytogenetic findings in FAB M2, M4 and other FAB groups Comparing frequency of normal versus complex, respectively normal versus abnormal karyotypes in M2 and M4 cases we observed a significantly lower presence of normal karyotypes in the M2 class (p=0,013 when compared to KTC, respectively p=0,022 when compared to abnormal karyotypes). Morphologic dysplastic features within FAB classes are depicted in table 6. Table 6. Morphologic dysplastic features within the FAB classes FAB M0 M1 M1/2 M2 M3 M2/4 M4 M4/5 M5 M6 Mx Dys Dys Dys Dys There were no differences regarding dysplasia between M2 and M4 classes when presence or absence of dysplasia was compared (p=0, 76), nor when multilineage dysplasia (Dys 2 + Dys 3) was compared to the number of cases with dysplasia on at least one lineage (Dys 0 + Dys 1) (p=0, 37). (Figure 2) Figure 2. Morphologic dysplasia in M2, M4, other FAB classes 140

7 Next we compared dysplasia among cytogenetic subgroups with normal karyotype versus complex, respectively abnormal karyotype. There was no significant difference whether we compared presence /absence of dysplasia or multilineage dysplasia versus at least one lineage dysplasia. (Figure 3) Figure 3. Dysplasia compared to karyotype findings Dys0 / Dys1 + Dys2 + Dys3 in NKT / CKT p=0,76 Dys0 / Dys1 + Dys2 + Dys3 in NKT / abnormal KT p=0,47 Dys0 + Dys1 / MLD (Dys2 + Dys3) in NKT / CKT p=1 Dys0+ Dys1 / MLD (Dys2 + Dys3) in NKT / abnormal KT p=1 Nor presence or absence, neither degree of dysplasia correlated with the prognostic groups (p=0, 24) (Table 6). Last but not least we compared some biologic and hematologic parameters at diagnosis between following groups: MRC-AML as common group and separated into cmrc and mmrc, NOS-AML, a new group created by adding mmrc-aml to NOS- AML. Table 6. Dysplasia compared to prognostic groups Favorable Intermediate Negative Dys Dys Dys Dys Table 7. Biologic and hematologic parameters in AML classes constituted according to presence/ absence of cmrc, mmrc Age WBC (years) (x10 9 /l) Hb (g/dl) Plt (x10 9 /l) %Bl Sex ratio cmrc 55.6 ± ± ± ± ± B / 10F mmrc 49.6 ± ± ± ± ± B /6 F p 0,1273 0,1428 0,4438 0,6457 0,4920 0,7388 R squared 0,0652 0,0702 0,0190 0,0069 0,0159 MRC 52.9 ± ± ± ± ± F / 16 B NOS 53.2 ± ± ± ± ± F / 16 B p 0,9439 0,0001 0,6327 0,3133 < R squared < ,2076 0,0035 0,0156 0,

8 mmrc 49.6 ± ± ± ± ± B / 6 F NOS 53.2 ± ± ± ± ± B / 16 F p < R squared < cmrc 55.6 ± ± ± ± ± B / 10F NOS 53.2 ± ± ± ± ± B / 16 F p < R squared cmrc 55.6 ± ± ± ± ± B / 10F NOS+ mmrc 52.1 ± ± ± ± ± B / 22F p R squared In between the MRC group and subgroups there were no differences, but MRC group and each of its subgroups differed from AML-NOS by leukocyte count and blast percentage, but not by age, hemoglobin or platelet number. If the new constituted group mmrc+ NOS was compared to cmrc, differences regarding leukocytes and blasts were still present and significant (Table 7). Discussion In our group cytogenetic changes were present in 48% of the patients, in the low range if compared to most of the reports, where the frequency lies between 50 and 60% (Marchesi et al, 2010). Although it is presumed that there are some geographical and ethnical differences regarding the distribution of genetic aberrations in cancer patients generally and in acute leukemia (AL) particularly, these were insufficiently and inconsistently documented (Johansson et al., 2011), (Nakase et al, 2001). Even though there are certain differences in our group compared to previously reported prevalence, is is difficult to interpret them due the small number of patients in our group. As long as we cannot compare to cumulative data in our geographic region, conclusions cannot be drawn because even in Europe there are differences between regions. In our group we had two cases of AML with t(8;21)(q22;q22), representing 2,52%, less than in other reports in literature (5-10%) (Reikvam et al., 2011). On a series of 1897 cases of adult patients the prevalence was 4,6% (Schoch et al., 2003), less for older patients (Grimwade, 2001). Our cases are two young men (36 and 43 years old) (Table 8), without prior hematological disease or other malignant disease, without exposure to chemotherapeutic agents or benzene, thus being cases of de novo AML. Table 8. General data for t(8;21) cases WBC (x 1000/µl) Hb (g/dl) Plt (x 1000/µl) FAB %Bl Case Case Case 1 presented as additional aberration the absence of the y chromosome. Association of y and AL or 142 MDS has been discussed especially taking into account that both are age related phenomenon (Wong et al 2008). It seems

9 that for myeloid proliferations, rather than for lymphoid ones, constant absence of chromosome y in all analyzable metaphases might be interpreted as part of the malignant process (Wiktor et el., 2011). The young age of our patient represents an argument to consider that in this case y is an aberration associated to the malignant process. Both cases had Auer bodies; case 1 had unilineage dysplasia and case two bilineage dysplasia (granulocytic and erythroid). The salmon-pink color of the cytoplasm considered specifically linked to this cytogenetic alteration was not described in our cases (Nakamura et al, 1997). From the 79 case of our study two of them were APL, representing 2.52%. The reported frequency of such cases, bearing the t(15;17)(q22;q12)(pml- RARA) abnormality is 4-9% (Bain, 2010) and on a series of 1897 patients it was 5,2% (Schoch et al, 2003). This is the only type of leukemia for which more studies have confirmed a higher prevalence in certain populations (Douer et al, 1996), such as South America, Hispanic population in the United States, but also in Spain compared to the rest of Europe (Chillon et al, 2001). Both our cases were de novo AML in men in the sixth decade of life (52 and 56 years), which presented with pancytopenia and the typical morphological aspect in bone marrow with promyelocytes with multiple Auer rods. Our group comprised 1 case of AML inv(16), in a 55 years old male. This represented 1.29% of all cases, less than mentioned by other studies (3-8%) and less then in Schoch s series (4.5%). Our case presented with leukocytosis (25,000/ µl), anemia, thrombocytopenia (Hb 6g/ dl, Plt 40,000/µl) and typical myelomonocytic morphology with 40% blasts and 42% eosinophils, morphological abnormal in all maturational stages, presenting proeosinophylic granules and mature hyolobulated eosinophils. In our series we had 3 cases with trysomy 8 as sole abnormality, one case wuth +8 and +12 and one case in which trysomy 8 was part of a complex karyotype. Although high frequency of trysomy 8 in AML cases is recognized, clinical biological relevance could not been demonstrated even on great studies and it is considered to be rather a secondary event, than a primary cytogenetic or molecular event (Schoch et al, 2006). In our group trysomy 8 cases were heterogeneous without any common characters. Assignment to cytogenetic prognosis classes meant attributing to 6.33% of the cases favorable prognosis, while 27.84% of the cases had a negative prognosis, the rest of them (65.82%) being defined as intermediate risk cases. All studies also have a higher proportion of intermediate cases, probably because of the high rate of normal karyotype but these cases can be further classified by molecular biology (FLT3, NPM1, CEBPA). Frequency of dysplastic findings in our series was close to other studies data (shown in table 9): Table. 9 Frequency of dysplasia in de novo AML cases (literature review adapted from Wandt et al, 2008) Author Study, year Number of patients Dys 0 (%) DysG (%) DysE (%) DysM (%) Dys2 (%) Dys3 (%) Brito-Babapulle MRC 1987 et al Jinnai et al Estienne et al Lille, Goasguen et al ECOG, Goasguen et al AMLCG Ballen et al Kuriyama et al

10 Gahn et al Meckenstock et 1997 al Kahl et al Kuriyama et al JALSG, Haferlach et al AMLCG-92, Arber et al Miyazaki et al Wandt et al without dysplasia 32.47% (7-57.6%), granulocytic series dysplasia 51.95% (7-54.8%), erythroid series dysplasia 45.45% ( %), megakaryocytic series dysplasia 15.58% ( %), triliniar dysplasia 10.39% (7.8-26%). The composition of our group allowed comparison between M2 and M4 cases. We did not find any differences regarding dysplasia (present/ absent/ number of affected lineages), but for M2 frequency of abnormal karyotypes was significantly higher. In spite of these findings, comparing cases with complex or pathologic karyotype to cases with normal karyotype there were no significant differences regarding dysplasia, this might be due to the extreme heterogeneity of non M2,non M4 cases, which thus modified proportions when taken into account. On our study there was no correlation between cytogenetic risk classes and presence of dysplasia, as opposed to the study of Haferlach et al (2003), which showed a higher frequency of patients without dysplasia in the favorable prognostic group and also a higher frequency of trilineage dysplasia in the unfavorable prognostic group, although the same study did not show different overall survival means between the group with MLD and the other cases. Because one of the subjects of debate is whether multilineage dysplasia should be used as classification tool, we decided to compare cases with multilineage dysplasia with other types of cases. After we excluded cases with recurrent cytogenetic abnormalities, we 144 constituted following groups: cmrc-aml, mmrc-aml, MRC-AML (c+m), mmrc + NOS AML. We compared biological and hematological parameters at diagnosis and identified significant differences for leukocyte count and blast percentage between AML-NOS and MRC-NOS, whether these were considered as common group or two distinct subgroups (c and m). When we added the mmrc group to the NOS and compared this newly created group to cmrc, the differences for the two above mentioned parameters were still present, although the difference of the means was less. If we admit that leukocyte count and blast percentage at diagnosis represent prognostic factors, this would mean that AML-NOS presents at onset more frequently associated risk factors than MRC-AML. On the basis of our result we can also affirm that at onset some hematological parameters are resembling between mmrc and cmrc, but differ in AML-NOS. Comparison of such groups has been made until now mostly regarding prognostic indicators like achievement of complete remission (CR), overall survival (OS) and event free survival (EFS). Some of these studies showed multilineage dysplasia as to be relevant (Gahn et al, 1996) as prognostic factor, others did not confirm this hypothesis. (Kahl et al, 1997) Morphological studies of Arber on 30 patients showed reduced OS in patients with MLD, without any differences between AML with MDS history and AML de novo with MLD, but critique has been brought to this study that the control group comprised cases with recurrent genetic alterations which generally show good

11 prognosis (Arber et al, 2003). Weinberg et al (2009) analyzed 100 patients and found lower OS and CR in patients with MRC- AML compared to AML-NOS, but did not study the cases with cmrc and with MLD separately (Weinberg et al., 2009). A more detailed analysis has been done by Miesmer and colleagues on 408 cases. They did not found differences on survival between cases with/ without dysplasia. In contrast with our results they found a much greater frequency of dysplastic changes in the cmrc group. They did not find differences regarding overall survival between patients with MLD and all the other ones, selected or unselected on the basis of normal/pathological karyotype. In contrast with our results, patients with MLD were younger than NOS patients, and leukocyte number at diagnosis showed no significant difference. CMRC patients had worse prognosis indicators, were older and had lower leukocyte counts than patients with MRC and NOS. In Miesmers study the group constituted of patients with NOS+MLD were younger, had higher leukocyte counts and better OS and EFS. Most relevant and recent studies consider that MLD, even if correlated with recognized adverse prognosis factors (i.e. unfavorable karyotype), does not have a direct and independent influence on prognosis, when more parameters are added in multivariate analysis. Thus even if our results suggest that both MLD and cmrc correlate with higher leukocyte count and blast percentage, we have to admit that for each of the two groups there might be additional and more important prognosis factors, which give the final touch. Most probably these factors are at molecular level, some of them being already recognized (FLT3, CEBPA, NPM1) (Falini et al, 2010). Conclusions Quantification of dysplasia according to WHO criteria is a process subjected to high degree of subjectivism and to a series of technical impediments. 145 The number of analyzable cells is reduced if smears are hypocellular, with crushed elements or with high blast percentage. Up to date the significance of assessing dysplasia has not yet been demonstrated. The WBC and blast percentage have always been recognized as prognostic factors, even if not always as independent prognostic factors. This means that our observations regarding the correlations of these parameters with MRC cannot be neglected. Inclusion of one particular patient into a cytogenetic prognostic group is only a process of risk- benefit optimization in choosing therapy. Although our experience is reduced as number of cases, it is obvious that, at current knowledge, the outcome for each particular case is influenced by a summum of factors and only complete investigations (morphology, immunophenotyping, cytogenetics and molecular biology) and correlation of these data may explain therapy responses that do not correlate with the cytogenetic risk group pattern. References Arber, D.A.; Stein, A.S.; Carter, N.H. : Prognostic impact of acute myeloid leukemia classification. Importance of detection of recurring cytogenetic abnormalities and multilineage dysplasia on survival. Am. J. Clin. Pathol., 119, 5, , 2003 Bain, J.B.: Leukemia Diagnosis. 4 th edition. John Wiley et Sons, 2010 Bennett, J.M.; Catovsky, D.; Daniel, M.T. Proposals for the classification of the acute leukaemias. French-American-British (FAB) co-operative group". Br. J. Haematol. 33, 4, , Bennett, J.M.; Catovsky, D.; Daniel, M.T. Proposed revised criteria for the classification of acute myeloid leukemia. A report of the FAB cooperative group. Ann Intern Med, 103, , 1985 Chillon, C.M.; Garcıa-Sanz R.; Balanzategui, A.; Ramos,.; Fernandez-Calvo, J.; Rodrıguez, M.J.; Rodrıguez-Salazar, M.I.; Corrales, A.; Calmuntia, M.J.; Orfao, A.;, Gonzalez, M.; San Miguel, J.F.:. Molecular characterization of acute myeloblastic leukemia according to the new WHO classification: a different

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