Presence Of Multilineage Dysplasia Research Articles

Clinical Outcomes and Mutational Profile Associated with Dysmyelopoiesis in Chronic Myelomonocytic Leukemia

Chronic myelomonocytic leukemia (CMML) is defined by absolute (≥ 0.5 x10 9/L) and relative (≥ 10%) monocytosis in peripheral blood. Presence of significant dysplasia in any of the three myeloid series, in particular dysgranulopoiesis, is an almost constant finding in CMML. In the absence of a clonal marker, a scenario very unlikely with the advent of next-generation sequencing (NGS), significant myeloid dysplasia becomes a crucial criterion for establishing the diagnosis. Although bone marrow dysplasia has demonstrated independent prognostic value in MDS, its significance in relation to CMML has never been assessed. The main interest of our work was to evaluate the genomic determinants of dysplasia and to assess its prognostic impact. We analyzed a series of 240 patients with CMML with clinical, morphological, immunophenotypic, cytogenetic and molecular information. Patients were diagnosed following WHO and ICC 2022 recommendations. As a novelty with respect to previous WHO classifications, the new definition of CMML includes those patients previously categorized as oligomonocytic CMML (OM-CMML). That is, myelodysplastic neoplasms (MDS) or unclassifiable myelodysplastic/myeloproliferative neoplasms with relative monocytosis and a monocyte count of 0.5 × 10 9/L to <1 × 10 9/L. A total of 167 (71%) patients were diagnosed with CMML-1 and 67 (29%) with CMML-2. Likewise, 193 (80%) patients were diagnosed with dysplastic CMML, 55 of whom would meet criteria for OM-CMML, and 47 (20%) were diagnosed with proliferative CMML. We assessed associations between dysmyelopoiesis and gene mutations by Spearman's rank correlation coefficient. Dysgranulopoiesis showed a significant positive correlation with NRAS, TET2 and ZRSR2 mutations; while a significant negative correlation with SETBP1 was observed. Dysmegakaryopoiesis showed a significant positive correlation with ASXL1 and TP53. Dyserythropoiesis showed a significant positive correlation with DNMT3A and SF3B1. The heatmap and matrix showing these correlations, as well as the differences in the degree of dysmyelopoiesis among these specific mutations are depicted in Figure 1-A & 1-B. Later, we wanted to assess the prognostic impact of dysgranulopoiesis, dyserythropoiesis and dysmegakaryopoiesis in terms of overall survival (OS) and leukemia-free survival (LFS) in our series. By applying the MaxStat package in R, it allowed us to detect the best cut-off points for dysgranulopoiesis (>75%) and dysmegakaryopoiesis (>20%) to separate our series into two groups with significantly different outcomes (Figure 1-C). We were unable to detect a significant cut-off point for dyserythropoiesis and the presence of multilineage dysplasia also showed no prognostic impact. CMML patients with dysgranulopoiesis above 75% showed a significant shorter median OS (34.4 vs 67.8 months; Hazard Ratio (HR): 1,99 [1.24-3.19]; P=0.0036) and LFS (33.4 vs 66.6 months; HR: 1,87 [1.17-3]; P=0.008). Likewise, patients with dysmegakaryopoiesis above 20% also showed a significant shorter median OS (30.2 vs 67.1 months; HR: 2.52 [1.63-3.88]; P<0.0001) and LFS (30.4 vs 66.6 months; HR: 2.76 [1.78-4.28]; P<0.0001). Remarkably, dysmegakaryopoiesis above 20% and dysgranulopoiesis above 75% were identified as independent prognostic factors for both OS and LFS after adjustment by multivariable Cox regression analyses by CMML-specific prognostic scoring system (CPSS) and Mayo Prognostic Model. Furthermore, the addition of dysmegakaryopoiesis above 20% and dysgranulopoiesis above 75% to these prognostic indices improved their prognostic accuracy in terms of OS as assessed by a C-index for right-censored data (Figure 1-C). In conclusion, our study has allowed us to detect mutations in genes related to dysmyelopoiesis. In addition, both dysgranulopoiesis and dysmegakaryopoiesis have demonstrated a negative prognostic impact on the survival of patients in our series that was not captured by the CPSS and Mayo prognostic model. Their worse prognosis could be partially justified by their association with poor prognostic mutations (e.g. NRAS, ASXL1, TP53), which allows us to visualize them as a good surrogate of poor prognostic molecular profiles.

Rapid fluorescence in situ hybridisation optimises induction therapy for acute myeloid leukaemia.

Rapid fluorescence in situ hybridisation optimises induction therapy for acute myeloid leukaemia.

Multilineage dysplasia as assessed by immunophenotype has no impact on clinical-biological features and outcome of NPM1-mutated acute myeloid leukemia

The presence of multilineage dysplasia (MLD) by morphology at diagnosis in acute myeloid leukemia (AML) defines a separate subset in the World Health Organization classification with still-debated prognostic value. A major controversy concerns MLD's role in NPM1-mutated (NPM1⁺) AML, which correlates with good prognosis. We used flow cytometry (FC), an emerging technique for assessing dysplasia, to investigate MLD in NPM1⁺ AML by an immunophenotypic score (IPS), a technique previously adopted in myelodysplastic syndrome. Eighty-five intensively treated NPM1⁺ AML cases were studied. Patients were grouped according to the combination of data in maturing cell compartments. FC-assessed dysplasia showed a significant correlation with morphology-assessed dysplasia, showing the efficacy of this method in highlighting dysplasia in AML. Except for MLD, IPS did not influence any patient- or disease-related characteristics at diagnosis. Furthermore, IPS did not influence complete remission rate, disease-free survival, or overall survival. By investigating NPM1 status on separated cell compartments, we established a correlation between FC-assessed MLD and belonging to AML clone. This study shows that dysplasia evaluated by immunophenotype has no impact on clinical-biological characteristics or on outcome of NPM1⁺ AML. Dysplasia is part of the spectrum of NPM1⁺ AML, and the prognostic stratification of this category of patients should not be based upon it.

Role of ASXL1 and TP53 mutations in the molecular classification and prognosis of acute myeloid leukemias with myelodysplasia-related changes.

Acute myeloid leukemias (AML) with myelodysplasia-related changes (AML-MRC) are defined by the presence of multilineage dysplasia (MLD), and/or myelodysplastic syndrome (MDS)-related cytogenetics, and/or previous MDS. The goal of this study was to identify distinct biological and prognostic subgroups based on mutations of ASXL1, RUNX1, DNMT3A, NPM1, FLT3 and TP53 in 125 AML-MRC patients according to the presence of MLD, cytogenetics and outcome. ASXL1 mutations (n=26, 21%) were associated with a higher proportion of marrow dysgranulopoiesis (mutant vs. wild-type: 75% vs. 55%, p=0.030) and were mostly found in intermediate cytogenetic AML (23/26) in which they predicted inferior 2-year overall survival (OS, mutant vs. wild-type: 14% vs. 37%, p=0.030). TP53 mutations (n=28, 22%) were mostly found in complex karyotype AML (26/28) and predicted poor outcome within unfavorable cytogenetic risk AML (mutant vs. wild-type: 9% vs. 40%, p=0.040). In multivariate analysis, the presence of either ASXL1 or TP53 mutation was the only independent factor associated with shorter OS (HR, 95%CI: 2.53, 1.40-4.60, p=0.002) while MLD, MDS-related cytogenetics and previous MDS history did not influence OS. We conclude that ASXL1 and TP53 mutations identify two molecular subgroups among AML-MRCs, with specific poor prognosis. This could be useful for future diagnostic and prognostic classifications.

Characteristics of acute myeloid leukemia with myelodysplasia‐related changes: A retrospective analysis in a cohort of Chinese patients

We retrospectively analyzed 449 patients with AML under the WHO classification of AML 2008 and probed implications of this classification in diagnosis and treatment of acute myeloid leukemia with myelodysplasia-related changes (AML-MRC) among them. The clinical presentations, biological features, treatments, and prognosis of patients diagnosed with AML-MRC were analyzed and compared with those of AML not otherwise specified (AML-NOS). In all patients, 115 (25.6%) were diagnosed as AML-MRC including 64 males and 51 females with median onset age of 48 years (range from 17 to 78). Their complete remission (CR) rate was 60.9% and relapse rate was 57.1%. The observed median overall survival (OS) and disease-free survival (DFS) were 10 and 5 months, respectively, which was significantly shorter than those of AML-NOS patients (P < 0.05). The prognosis of AML-MRC patients with myelodysplastic syndrome (MDS)-related cytogenetics sole was similar to those with history of MDS or myelodysplastic/myeloproliferative neoplasm (MDS/MPN). Patients with MDS-related cytogenetic abnormalities and/or history of MDS or MDS/MPN predisposed significantly shortened CR, OS, and DFS than AML-MRC patients with only multilineage dysplasia (MLD) and AML-NOS patients (P < 0.05). Multivariate analysis showed that age, cytogenetics, and history of MDS or MDS/MPN were independent prognostic factors. Patient diagnosed as AML-MRC presented distinctive clinical and biological features. Presence of MLD does not change the prognosis.

Multilineage Dysplasia Confers Poor Prognosis to Patients with De Novo Acute Myeloid Leukemia with Intermediate-Risk Cytognetics and Wild-Type NPM1.

AbstractAbstract 2551▪▪This icon denotes a clinically relevant abstract Background:The presence of multilineage dysplasia (MLD) is the hallmark to define acute myeloid leukemia (AML) with myelodysplasia-related changes. The recognition by the WHO classification of this category as a separate entity implies the presence of differential biological and clinical features. While this seems to be confirmed for patients with AML and MLD associated with high risk cytogenetic abnormalities, the prognostic significance of MLD in cases with intermediate risk karyotype is unclear. Recent studies have shown that MLD has no prognostic impact in patients with mutated NPM1, but the prognosis of patients with intermediate-risk cytogenetics and MLD harboring a wild-type configuration of NPM1 is uncertain. Objective:The aim of this study was to analyze the presence of MLD and its prognostic value in AML patients with intermediate risk cytogenetics (IR-AML) and wild-type NPM1. Patients and methods:One-hundred eighty-two patients diagnosed with de novo IR-AML and wild-type NPM1 treated with the same chemotherapy protocol (CETLAM-2003) were included (M/F:101/81; median age: 55.5, 18–73). Bone marrow aspirate smears performed at diagnosis were reviewed by a panel of expert cytologists, and the cases were categorized as having MLD by applying strictly the WHO criteria (Harris et al, WHO 2008, MLD-1). Additionally, cases with dysplasia in ≥50% of the cells in one cell line, and between 30% and 50% in another cell line, were classified as having significant dysplasia (MLD-2). Results:The degree of dysplasia was evaluable in 139 of the 182 cases, observing strictly WHO-defined multilineage dysplasia (MLD-1) in 45 (25%), significant dysplasia (MLD-2) in 17 additional patients (9%), and absence of MLD in 80 (44%). In 43 (30%) cases dysplasia could not be thoroughly quantified in all hematopoietic lines, mostly due to the lack of enough evaluable precursors in a context of massive blast infiltration. No major differences concerning main characteristics were observed between patients with or without MLD, including age, leucocyte count at presentation, degree of BM involvement, proportion of normal karyotype, and frequency of mutations of FLT3 (internal tandem duplication), CEBPA, and partial tandem duplication of MLL gene. Since the outcome of patients with MLD-2 was similar to that of patients with stringent WHO MLD, and markedly inferior to patients lacking MLD (table 1), these two groups were merged in a unique MLD group (MLD-1+MLD-2). Thus, MLD was associated with a lower probability to achieve complete remission (CR) after frontline induction chemotherapy (66% vs. 80%, p=0.033). In addition, patients with MLD showed a trend to an inferior overall survival (OS at 5 years: 39.2±5% vs. 22.9±8.1%, p=0.064). Of note, a multivariate analysis identified increasing age, higher leucocyte count at presentation, and presence of MLD (hazard ratio, 95% CI: 1.569,1.056–2.331; p=0.026) as the only variables associated with a shortened survival; this analysis also included presence of FLT3-ITD and cytogenetics (normal karyotype vs. other intermediate-risk abnormalities). Age was the only variable with independent prognostic value on leukemia-free survival in this cohort of patients, whereas MLD did not reach statistical significance. Interestingly, the outcome of patients with MLD-AML with a minimum CR duration of 3 months was significantly better after allogeneic stem cell transplantation in first CR compared to other post-remission treatments (5-year OS: 82±12% vs. 31±9%, p=0.006), suggesting that alloHSCT could partially overcome the adverse prognosis associated with this AML category.Table 1Medians for survival time (months)95% CIEstimateSELower boundUpper boundNo MLD35.2008.19219.14451.256MLD120.5334.15212.39628.671MLD24.5336.7000.017.665 Conclusions:The presence of MLD confers adverse prognosis to patients with IR-AML and wild-type NPM1. Presence of significant dysplasia in two hematopoietic cell lines without fulfilling stringent WHO criteria was associated to an outcome similar to that of patients with WHO-defined criteria, an observation that leads to a wider interpretation of significant MLD in AML. These results have an important clinical relevance and should lead to the search of new genetic and epigenetic markers associated with MLD in this setting.This study has been partially supported by grant n° 03/0423 from Instituto de Salud Carlos III/FIS0. Disclosures:No relevant conflicts of interest to declare.

Multilineage Dysplasia Has No Prognostic Impact in 108 Adult Patients with CEBPA Mutated AML Supporting the WHO Proposal to Classify These Patients As A Molecularely Defined Unique Entity

Abstract 1443 Background:In the 2008 WHO classification, CEBPA and NPM1 mutated AML are considered as “provisional entities”. However, patients without “recurrent genetic abnormalities” are classified as “AML with MDS-related changes, AML-MRC” based on multilineage dysplasia (MLD), cytogenetics, clinical history, or “AML, not otherwise specified”. MLD+ patients may also carry CEBPA mutations but will still be classified as AML-MRC. Study Design: We investigated the prognostic impact of MLD in 108 CEBPA mutated AML (54 males, 54 females; median age 67.2; range 15.7–87.6 years) who were all characterized by cytogenetics and investigated for molecular markers in subcohorts. MLD was defined by ≥50% dysplastic cells in 2–3 hematopoietic lineages according to WHO. CEBPA mutation analysis was done by Sanger sequencing or deep-sequencing (454 Life Sciences, Branford, CT). 44 pts (40.7%) had mono-, 53 (49.1%) biallelic, and 11 (10.2%) homozygous CEBPA mutations. Frequencies of other markers were: FLT3 -ITD: 10/105 (9.5%); FLT3 -TKD: 2/79 (2.5%); NPM1: 15/105 (14.3%); MLL -PTD: 4/105 (3.8%); IDH1/2: 8/63 (12.7%); RUNX1: 8/90 (8.9%). Results: Dysplasia (≥50% of cells) was detected in granulopoiesis in 46/106 cases (43.4%), in erythropoiesis in 14/108 (13.0%), and in megakaryopoiesis in 34/90 (37.8%). MLD was found in 28/108 (25.9%) of bone marrow samples: two-lineage dysplasia in 26, trilineage dysplasia in 2 cases. According to MRC criteria (Grimwade et al., Blood, 2010), the majority had intermediate karyotypes (KTs) (101/108; 93.5%) mainly due to normal KTs (NK; n=80/101). This confirmed the strong association of CEBPA mutations with NK-AML. Adverse KTs were identified in 7 pts only (6.5%). MLD+ and MLD- CEBPA mutated pts had similar median age (MLD+: 68.7; MLD-: 66.7 yrs; p=n.s.) and male/female ratios (1.5 vs. 0.9; p=n.s.). MLD+ had lower mean WBC counts (13.7 vs. 38.6×10(9)/l; p=0.004), but mean platelet counts, Hb, and BM blasts did not differ significantly from MLD-. The frequency of MLD+ did not differ significantly between monoallelic (14/45; 31.1%), biallelic (11/53; 20.8%), or homozygous CEBPA mutations (3/10; 30.0%). Intermediate and adverse KTs were similarly distributed between MLD+ and MLD- cases (intermediate: 27/28; 96.4% vs. 74/80; 92.5%; adverse: 1/28; 3.6% vs. 6/80; 7.5%; p=n.s.). Also, additional NPM1 mutations showed a similar frequency in MLD+ (4/28; 14.3%) or MLD- (11/77; 14.3%) cases, which holds true as well for FLT3 -ITD (frequency in MLD+: 1/28; 3.6%; in MLD-: 9/77; 11.7%; p=n.s.) or FLT3 -TKD, RUNX1, MLL -PTD, IDH1/2, or NRAS mutations. Outcome did not differ significantly between MLD+ and MLD- (2-year OS rate: MLD+: 56.5%; MLD-: 65.5%; median EFS: MLD+: 13.8 months; MLD-: 16.3 months, p=n.s.). OS and EFS were independent from mono-, biallelic, or homozygous CEBPA mutations in the total cohort, but when only FLT3 -ITD negative pts were considered, biallelic as compared to monoallelic/homozygous CEBPA mutations had better median OS (not reached, n.r. vs. 23.3 months; p=0.060) and median EFS (22.8 vs. 14.4 months; p=n.s.). Adverse KTs had worse OS than intermediate KTs (median 8.4 months vs. n.r.; p=0.021), while EFS was not significantly influenced by MRC group. By univariable Cox analysis, gender (better for female; p=0.024), age (p=0.031), WBC count (p=0.003) (continuous), and MRC risk category (p=0.03) were prognostically relevant for OS, whereas presence of MLD, Hb, platelets, BM blasts (continuous), biallelic vs. monoallelic/homozygous CEBPA mutations, FLT3 -ITD, and NPM1 mutations had no impact on survival. MLD also had no significant impact on EFS in univariable analysis. By multivariable analysis, only male sex (p=0.01), higher WBC count (p<0.001), and unfavorable MRC category (p=0.045) had significantly shorter survival. Multivariable analysis showed an adverse impact on EFS for gender (p=0.014) and high WBC count (p=0.003). Conclusion: Presence of MLD has no impact in pts with CEBPA mutated AML with respect to biological characteristics, cytogenetic risk group, associated molecular markers, and prognosis. Thus, our data does not support to overrule the detection of CEBPA mutations for classification and prognosis by the presence of MLD as suggested by the WHO guideline. We therefore strongly suggest to categorize CEBPA mutated AML as a separate entity and further delineate patients only according to mutation status and additional FLT3 -ITD but not by dysplasia. Disclosures:Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Macijewski:MLL Munich Leukemia Laboratory: Employment. Grossmann:MLL Munich Leukemia Laboratory: Employment. Kohlmann:MLL Munich Leukemia Laboratory: Employment. Alpermann:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

Long-Term Outcome of Anemic Non Del 5q Lower-Risk MDS Refractory to or Relapsing After Erythropoiesis Stimulating Agents (ESAs)

Abstract 442 Background. ESAs are usually the first line tx of anemia in non del 5q lower risk MDS. However, not all pts respond to ESAs and median response duration is only about 2 years (Park, Blood 2008;111:574). Long-term outcome of pts who do not respond to or relapse after response to ESAs is incompletely known. We analyzed this outcome by updating a previously reported lower-risk MDS cohort of 403 pts treated with ESA in centers of the GFM (Blood 2008;111:574). Methods. We analyzed in that cohort low and int-1 (lower risk) IPSS pts with Hb Results. 177 pts, including 94 with primary resistance to ESAs and 83 with relapse after an initial response were analyzed. In the 94 pts with primary resistance to ESAs, M/F was 2, median age 75, WHO classification at tx onset RA, RCMD, RARS, RAEB-1 in 17%, 27%, 28%, 27% of cases, respectively (resp), karyotype fav, intermediate (int) in 86% and 14% pts, resp, IPSS low, int-1 or assignable to low/int-1 in 35%, 60% and 5% pts, resp. Median serum ferritin was 658 ng/mL and median serum EPO level (sEPO) 125 IU/L. 63% of the pts were RBC transfusion dependent (TD) (median 2 RBC units/month). Median overall survival (OS) and 3-y cumulative incidence (CI) of AML from tx onset were 43 months (mo) and 18%, resp. Among pre-tx characteristics, age >75 was associated with shorter survival (median OS 31 mo vs. not reached for age 65 (P=0.93), and their median OS was not reached vs 41 mo in pts aged >65 (P=0.03). 83 pts relapsed after an initial response (IWG 2000 major and minor in 60.2% and 39.8% pts, resp) of 16.5 mo median duration (range 3–74 mo). At tx onset, M/F was 1.35, median age 74.3, WHO classification RA, RCMD, RARS, RAEB-1 in 14%, 38%, 32%, 16% of cases, resp, karyotype fav, int in 92% and 8% pts, resp, IPSS low, int-1 in 51% and 49% of pts. Median serum ferritin was 695 ng/mL and median sEPO 64 IU/L. 45% of the pts were TD (median 2 RBC units/mo). Median OS and 3-y CI of AML after relapse were 53 mo and 9.7%, resp. Median OS after relapse was 26 mo in RAEB-1 and not reached in other WHO subtypes (P=0.06) and was not influenced by the presence of multilineage dysplasia. Pts who relapsed after 24 mo had a 4.1% 3-y CI of AML vs 12.8% in pts who relapsed before 24 mo (P=0.40). Median OS was not reached in pts who relapsed after 24 mo vs 53 mo in those relapsing before 24 mo (P=0.90). No pre-tx characteristic was predictive of relapse before or after 24 mo. 16% of the 83 pts were aged 65 (P=0.17), and their 3- CI of AML after relapse was 0% vs 12% in pts aged >65 (P=0.31). In the overall pt population (ie pts with primary resistance, pts with relapse and pts with sustained response), univariate competing risk modeling found CI of death from cardiovascular causes to be correlated with TD and older age at tx onset but not with response status, while only age remained significant in multivariate analysis (HR=1.12 [1.014-1.24], P=0.02). Both older age and early failure (ie primary failure or relapse Conclusions. Failure to respond or loss of response to ESAs in the absence of frank disease progression to AML or higher risk MDS was not associated with poor outcome in lower-risk MDS, except in some pt subgroups (pts with intermediate karyotype and with a diagnosis of RAEB-1). Those figures have to be taken into account for therapeutic decisions, especially in pts aged Disclosures: Off Label Use: ESAs for anemia in MDS. Fenaux: CELGENE, JANSSEN CILAG, ROCHE, AMGEN, MERCK, GSK, NOVARTIS, CEPHALON: Honoraria, Research Funding.

Multilineage dysplasia (MLD) in acute myeloid leukemia (AML) correlates with MDS-related cytogenetic abnormalities and a prior history of MDS or MDS/MPN but has no independent prognostic relevance: a comparison of 408 cases classified as “AML not otherwise specified” (AML-NOS) or “AML with myelodysplasia-related changes” (AML-MRC)

AbstractThe World Health Organization classification of acute myeloid leukemia (AML) is hierarchically structured and integrates genetics, data on patients' history, and multilineage dysplasia (MLD). The category “AML with myelodysplastic syndrome (MDS)–related changes” (AML-MRC) is separated from “AML not otherwise specified” (AML-NOS) by presence of MLD, MDS-related cytogenetics, or history of MDS or MDS/myeloproliferative neoplasm (MPN). We analyzed 408 adult patients categorized as AML-MRC or AML-NOS. Three-year event-free survival (EFS; median, 13.8 vs 16.0 months) and 3-year overall survival (OS; 45.8% vs 53.9%) did not differ significantly between patients with MLD versus without. However, MLD correlated with preexisting MDS (P < .001) and MDS-related cytogenetics (P = .035). Patients with MLD as sole AML-MRC criterion (AML-MLD-sole; n = 90) had less frequently FLT3 internal tandem duplication (P = .032) and lower median age than AML-NOS (n = 232). Contrarily, patients with AML-NOS combined with AML-MLD-sole (n = 323) had better 3-year EFS (16.9 vs 10.7 months; P = .005) and 3-year OS (55.8% vs 32.5%; P = .001) than patients with history of MDS or MDS/MPN or MDS-related cytogenetics (n = 85). Gene expression analysis showed distinct clusters for AML-MLD-sole combined with AML-NOS versus AML with MDS-related cytogenetics or MDS history. Thus, MLD alone showed no independent clinical effect, whereas cytogenetics and MDS history were prognostically relevant.

The Presence of Multilineage Dysplasia (MLD) Has No Significant Impact On Biological, Clinico-Pathological, and Prognostic Features in AML with Mutated Nucleophosmin (NPM1).

Abstract 2618▪▪This icon denotes an abstract that is clinically relevant.Poster Board II-594AML with mutated nucleophosmin (NPM1) is a provisional entity in the WHO-2008 classification of myeloid neoplasms. A yet unresolved issue concerning NPM1-mutated AML is the biological and clinical significance of concomitant multilineage dysplasia (MLD). So far, NPM1+ AML with MLD is classified as AML with myelodysplasia-related changes in the new WHO classification. Therefore, we addressed the impact of MLD in 378 de novo AML patients with mutated/cytoplasmic NPM1 and performed comparison of cytomorphological features, cytogenetics, the FLT3 mutation status, and gene expression profiles in patients with and without MLD. MLD - as defined by dysplastic features in at least two hematopoietic lineages by cytomorphology - was present in 95/378 (25.1%) cases. Mean age (±standard deviation) was 52.1±14.0 years in the group with MLD and 54.8±14.5 in the group without MLD (p=0.11). Males comprised 50/95 (52.6%) in the group with MLD vs. 119/283 (42%) in the group without MLD (p=0.075). Morphological subtypes according to FAB/WHO were available in 365 cases (96.5%) and did not differ significantly between both groups. The median WBC was 17.1 G/l (range: 0.9–211.0 G/l) in the group with MLD and 35.6 G/l (range: 0.5–600.0 G/l) in the group without MLD (p=0.0027). Absence of CD34 expression was observed in 60/74 (81.1%) and 172/212 (81.1%) cases of the groups with and without MLD, respectively (p=1.0). Cytogenetic data was available in all 378 cases: An abnormal karyotype was present in 15/95 (15.8%) cases with MLD and in 37/283 (13.1%) cases without MLD (p=0.49). Molecular analyses detected the FLT3-ITD in 16/91 (17.6%) cases tested with MLD and in 110/276 (39.9%) cases without MLD (p<0.001). FLT3-TKD mutations were found in 7/80 (8.8%) patients within the group with MLD and in 21/221 (9.5%) patients within the group without MLD, respectively (p=1.0). Moreover, NPM1-mutated AML with or without MLD demonstrated non-discriminative microarray gene expression profiles (Affymetrix HG-U133 Plus 2.0). The gene expression profiles, however, clearly differed between NPM1-mutated AML and NPM1-wildtype cases as control group, e.g. by detectable downregulation of CD34 and upregulation of the characteristic HOX gene signature. This indicates that NPM1 mutations rather than MLD dictate the distinctive features of this AML subtype. With respect to clinical relevance, presence of MLD had no significant influence on overall survival (OS), however, it was related to a better event-free survival (EFS, median 33.3 vs. 13.9 months, p=0.0412), which is likely due to the fact that FLT3-ITD were less common in the MLD+ group (p=0.001). In addition also in our cohort the presence of an FLT3-ITD was associated with a significantly shorter EFS (median 11.2 vs. 22.3 months, p=0.0002) and a significantly shorter OS (median 12.5 months vs. not reached, p<0.0001). MLD also had no impact on outcome if analyzed within the groups with or without FLT3-ITD, respectively. Multivariate analysis revealed FLT3-ITD as the only parameter independently related to EFS (p=0.0051) and OS (p=0.0024). In conclusion, the discrimination of AML with MLD from AML without MLD in NPM1-mutated AML does not lead to distinct entities of AML since biologic features and outcome are largely similar. The FLT3-ITD mutation, however, again was confirmed to be an important biological parameter in AML with NPM1 mutations as demonstrated by a significantly worse outcome. Thus, our data underlines that NPM1+ AML is a distinct entity and further suggests that NPM1+ AML should be considered as one group irrespective of the presence or absence of MLD. Disclosures:Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership. Macijewski:MLL Munich Leukemia Laboratory: Employment. Weiss:MLL Munich Leukemia Laboratory: Employment. Schnittger:MLL Munich Leukemia Laboratory: Equity Ownership. Kern:MLL Munich Leukemia Laboratory: Equity Ownership. Kohlmann:MLL Munich Leukemia Laboratory: Employment. Miesner:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership. Falini:Xenomics: Patents & Royalties.

AML with Multilineage Dysplasia (MLD) Correlates with MDS-Related Cytogenetic Abnormalities and a Prior History of MDS or MDS/MPN but Has No Independent Prognostic Relevance: A Comparison of 461 Cases Classified as “AML Not Otherwise specified” or “AML with Myelodysplasia-Related changes” as Defined by the 2008 WHO Classification.

Abstract 823▪▪This icon denotes an abstract that is clinically relevant.The new WHO classification of AML first considers genetic and molecular findings, the history of the patient but also stresses the importance of multilineage dysplasia (MLD) as independent parameter: Cases not classified as AML with recurrent genetic abnormalities or therapy-related AML are classified as AML with myelodysplasia-related changes (MRC) with one of the following parameters: AML after MDS or MDS/MPN (s-AML), AML with MDS-related cytogenetics and/or AML with MLD. All other cases are classified “AML, not otherwise specified” (NOS). We analyzed 1740 AML, 12.5% were AML with recurrent cytogenetic abnormalities, 7.4% were t-AML. We then evaluated a subgroup of 461 pts with AML without recurrent cytogenetic abnormalities. All of them were included based on complete availability of clinical data, cytogenetic and molecular features and adequate material for morphologic assessment. After morphologic analysis another 53 cases had to be excluded as dysplastic features were not evaluable following WHO guidelines. Following the WHO proposal cases of the provisional entities “AML with mutated NPM1” and “AML with mutated CEBPA” have not been excluded. We investigated dysplasia in all cases (n= 408) and found 184 AML with MRC (45.1%) and 224 AML NOS (54.9%). In 93/184 (50.5%) cases MLD alone led to the classification as AML with MRC. Out of the remaining 91 cases 42 had AML arising from previous MDS or MDS/MPN and 55 had AML with an MDS-related cytogenetic abnormality (both parameters present in 5 cases). Event-free survival (EFS) and overall survival were comparable in AML with and without MLD (median EFS 13.8 vs. 15.2 months, n.s.; median OS 24.5 vs. not reached, n.s.). MLD was positively correlated to the incidence of pre-existing MDS or MDS/MPN (frequency in cases with pre-existing MDS or MDS/MPN vs. others 66.7% vs. 33.1%, p<0.0001) and to MDS-related cytogenetic abnormalities (frequency in cases with MDS-related cytogenetic abnormalities vs. others 54.5% vs. 33.7%, p= 0.004). Thus, to analyze whether or not MLD as a sole criterion has clinical impact the following evaluations focused on the subgroup AML MLD sole (AML with MLD and without other MRC in comparison to AML NOS regarding clinical and prognostic parameters including age, WBC, cytogenetics, molecular markers, OS and EFS. Median age was 61.7 vs. 66.0 years in AML MLD vs. AML NOS (range 20.4-85.0 vs. 18.3-88.1 years, p=0.027) and the median WBC count was 18.3 vs. 13.4 G/l (range 0.4-370.0 vs. 0.14-600.0 G/L, n.s.). Pts with AML MLD exhibited a slightly lower frequency of NPM1 mutations (58.0% vs. 64.7%, n.s.) and a lower frequency of FLT3-ITD (18.0% vs. 28.5%, p= 0.061), and showed slightly more frequently NRAS mutations (18.2% vs. 9.6%, n.s.). Frequencies of MLL-PTD and CEBPA-mutations were similar between both groups. In the subgroup AML MLD sole (AML with MLD and without other MRC) in comparison to AML NOS, the presence of MLD has no impact on EFS (median 17.5 vs. 16.0 months, n.s.) and OS (3-year-OS 54.8% vs. 53.9%, n.s.). Further analyses were performed for the total cohort (n=408). MDS-related cytogenetics was associated with shorter EFS (median 7.2 vs. 15.2 months, p=0.011) and OS (median 17.4 months vs. not reached, p=0.010). Also, s-AML was associated with shorter EFS (12.7 vs. 15.6 months, p=0.099) and OS (15.0 months vs. not reached, p=0.027). Furthermore we assessed the prognosis of pts with AML NOS combined with the subgroup of AML MRC showing MLD only (n= 317) vs. all other cases with AML MRC being assigned to this group based on cytogenetics or preceding MDS/MPN (n= 91). A significant difference in OS (AML NOS and AML MLD only vs. AML MRC based on cytogenetics or preceding MDS/MPN, median OS not reached vs. 15.8 months, p= 0.001) and EFS (median 16.8 vs. 10.2 months, p= 0.004) was seen. Among the other prognostic parameters, only absence of NPM1 mutation, presence of MLL-PTD and higher age were significantly associated with a worse EFS and OS. In multivariate analysis a higher age was the only parameter independently related to worse EFS (p<0.0001), and higher age (p<0.0001) and MLL-PTD (p=0.033) were the only parameters independently related to shorter OS. These facts lead to the conclusion that MLD alone has very limited clinical impact and that pts with MLD and without previous MDS or MDS/MPN or MDS-related cytogenetic abnormalities should not be classified only by morphology as AML MRC. We suggest that classification of pts with AML could be simplified by only using cytogenetics and prior history for a reproducible risk-stratification in the setting of clinical trials. Disclosures:Miesner:MLL Munich Leukemia Laboratory: Employment. Weiss:MLL Munich Leukemia Laboratory: Employment. Macijewski:MLL Munich Leukemia Laboratory: Employment. Schnittger:MLL Munich Leukemia Laboratory: Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership. Kern:MLL Munich Leukemia Laboratory: Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership.

The WHO's who of haematopoietic malignancies

The WHO's who of haematopoietic malignancies

Impact of transfusion dependency on survival in patients with early myelodysplastic syndrome without excess of blasts

We present a retrospective analysis of 137 patients with early MDS without excess of blasts that revealed transfusion dependency in 87% of the cases. A significant difference in overall survival was noted between patients receiving ≤2units and those receiving >2units of RBC transfusions/month (65.0 vs. 35.3 months, respectively, P=0.02). Univariate statistical analysis identified the presence of disease progression to advanced MDS (χ2=26.4, P=0.001) and the administration of >1 or >2units of RBC per month (χ2=15.9 and 14.6, respectively, P=0.001) as the most important parameters affecting survival. Nevertheless, even the administration of 1 RBC unit every 4–8 weeks had a significantly adverse impact on survival compared to non-transfused patients. Transfusion dependency itself did not affect disease progression as determined by the presence of multilineage dysplasia and adverse karyotype (expressed by the IM-1 or IM-2 score). Multivariate analysis confirmed disease progression towards leukemia as a highly significant independent variable affecting survival (P=0.0001). None of the other evaluated parameters had a significant impact on survival in patients with progressive disease. In non-transplanted patients without MDS progression, administration of >2units of RBC transfusions/month was the only independent variable with adverse impact on survival in patients with unilineage erythroid dysplasia (P=0.02). In patients with multilineage dysplasia, only heavy transfusion dependency (>3TURBC/month) and serum ferritin >2000μg/l adversely affected survival (P=0.03). Modification of the WPSS by replacing transfusion dependency with initial Hb level <80g/l retained its prognostic relevance and allowed the identification of a potential risk subset of early MDS patients with intermediate and high scores and limited survival (<40% at 5 years) as early as at the time of diagnosis. Our results confirm a significant negative impact of transfusion dependency on survival in patients with early MDS without excess of blasts. The main risk subgroup is characterized by unilineage dysplasia limited to erythropoiesis in combination with dependency on >2TU of RBC per month. These patients usually have prolonged survival that leads to the development of heavy transfusion iron overload and they thus represent the most important target group for intensive chelation therapy.

The Detection of Multilineage Dysplasia (MLD) Has No Influence on Prognosis in NPM1 Mutated Acute Myeloid Leukemia (AML) with Normal Karyotype

Acute myeloid leukemia with mutated nucleophosmin (AML NPM1mut) represents about one-third of all adult AML and shows distinctive biological and clinical features. For this reason, AML NPM1mut is planned to be included as a separate category in the revised WHO classification. A yet controversial issue, however, is whether AML NPM1mut with or without multilineage dysplasia (MLD) may differ biologically and clinically, as the presence of MLD might confer a negative prognostic impact. A further feature that was suggested to be typical for NPM1 mutated AML is “cup-like” morphology of blasts. We here analyzed 128 pts with AML NPM1mut and normal karyotype at first manifestation (59 females, 69 males; median age 60.5 years; 23.5–79.3 y). We investigated in parallel cytomorphology from bone marrow and/or peripheral blood, chromosome banding analysis, and molecular analyses. Presence of dysplasia was defined by dysplastic features in ≥50% of cells in the respective hematopoietic lineage as defined by the WHO. A 5% cut-off was taken for the presence of “cup-like” morphology of blasts. All cases were additionally analyzed for the FLT3-ITD, and in 122 pts for the FLT3-TKD. Statistical analysis was performed for overall survival (OS), and event-free survival (EFS) according to Kaplan-Meier using the 2-sided log-rank test. Cox regression analysis related OS and EFS with the analyzed parameters. We found a predominance of the FAB M1 (21.3% of all cases), M2 (33.9%), and M4 subtypes (28.3%). Cup-like morphology in ≥5% of all blasts was observed in 39 of 127 evaluable cases (31.3%) confirming previous observations of an association of the NPM1mut and this specific blast appearance. Molecular characterization detected NPM1 mutation subtype A (n=90/122; 73.8%), B (15/122; 12.3%), and D (7/122; 5.7%), which was in accordance to previous studies. In 56 cases (43.8%) there was a coincidence with an FLT3-ITD. Dysplasia of granulopoiesis was detected in 28/126 (22.2%), of erythropoiesis in 28/104 (26.9%), and of megakaryopoiesis in 57/87 (44.5%) cases in which the respective cell lineage could be analyzed. MLD (≥2 dysplastic hematopoietic lineages) was detected in 28 of 105 evaluable cases (21.9%). Clinical follow-up was available in 104 pts. (median follow-up 12,7 months). CR rate was 83.1% in 77 evaluable pts., and median EFS was 42.1 months in 104 evaluable pts (median OS not reached). An additional FLT3-ITD had a significantly inferior OS (p=0.003) and EFS (p=0.007), confirming the present series being representative. However, the presence of MLD was not significantly related to any endpoint such as CR rate, EFS, or OS. There was no association between MLD and the NPM1-subtype. Also, there was no significant correlation of MLD and the presence of a FLT3-ITD. In conclusion, the presence of MLD in AML NPM1mut with normal karyotype had no impact on CR rate and outcome, whereas coincidence of FLT3-ITD significantly worsened prognosis. These results give further evidence that AML with NPM1mut AML is a unique biological entity with clinical course mainly influenced by FLT3-ITD coincidence. These data do not support any additional prognostic influence of MLD in this AML subtype.

Clinical Characterization of Acute Myeloid Leukemia with Myelodysplasia-Related Changes as Defined by the 2008 WHO Classification System.

Background: Although some studies have validated the 2001 WHO classification of acute myeloid leukemia (AML), including the importance of multilineage dysplasia, others have suggested that multilineage dysplasia correlates with unfavorable cytogenetics but has no independent impact on prognosis. In 2008, the revised WHO classification system has expanded this category into “AML with myelodysplasia-related changes” (AML-MRC) that now includes 1) AML arising from myelodysplastic syndrome (MDS), 2) AML with MDS-related cytogenetic abnormalities, and 3) AML with multilineage dysplasia. An individual case may fall into this category by meeting any of the criteria. The goal of the current study is to clinically characterize this newly defined AML-MRC subgroup.Methods: One-hundred consecutive AML patients diagnosed at Stanford University Hospital between 2005 and 2007 with adequate material for mutation analysis were studied. Cases were classified using the 2008 WHO criteria. Diagnostic cytogenetic findings were reviewed and patients were stratified into risk groups using Southwest Oncology Group criteria. Available flow cytometry immunophenotyping results were reviewed and all samples were tested for NPM, FLT3 (ITD and D835) and CEBPA mutations. Clinical parameters including hemogram data at time of diagnosis were reviewed. Clinical follow-up including overall survival (OS), progression free survival (PFS) and complete remission (CR) rates were retrospectively determined. Kaplan-Meier methods and univariate and multivariate Cox proportional hazards regression analysis were used to compare the clinical data.Results: The cases included 57 males and 43 females with a median age of 56 (range 17–81). Cytogenetic risk-group stratification resulted in 9 patients with favorable, 65 with intermediate and 19 with unfavorable risk status. Using the 2008 WHO criteria, there were 48 AML-MRC, 40 AML not otherwise specified (AML-NOS), 9 AML with either t(8;21), inv(16) or t(15;17), and 3 therapy related AMLs. Overall, 26 patients had a NPM1 mutation (16 of which were FLT3 mutated), 25 had FLT3-ITD, 8 had FLT3-D835 and 9 had a CEBPA mutation (3 of which were FLT3 mutated). Compared to AML-NOS, patients with AML-MRC were significantly older (59 vs 51 years, p=0.014) and presented with lower hemoglobin (9 vs 11.2 g/dL, p=0.044), lower platelets (47 vs 54 K/uL, p=0.059), unfavorable cytogenetics (14/46 vs 3/36, p=0.014) and exhibited a decreased frequency of CEBPA mutation (0/46 vs 7/40, p=0.001) as compared to AML-NOS. Based on the flow cytometry immunophenotyping, the blasts from patients with AML-MRC more frequently expressed CD14 compared to AML-NOS (10/46 vs 4/36, p=0.048). Clinical outcome data showed that patients with AML-MRC had a significantly worse OS, PFS and CR compared to AML-NOS (Figure, all p<0.0001). Even after excluding the 14 patients with unfavorable cytogenetics from the AML-MRC group, the remaining patients with AML-MRC (defined solely by the presence of multilineage dysplasia) had worse outcomes compared to all AML-NOS patients (OS, p=0.013; PFS, p=0.012; CR, p=0.0076). Among 65 patients with intermediate risk cytogenetics, the outcome difference between the AML-MRC and AML-NOS groups remained significant (OS, p=0.0292; PFS, p=0.0232), also indicating prognostic significance of multilineage dysplasia. Within the AML-MRC group, univariate analysis showed that low platelets (<20,000/mm3), FLT3-D835 mutation and MDS-related cytogenetics correlated with OS (p=0.0456, p=0.0265, p=0.002 respectively) and PFS (p=0.0478, p=0.0626, p=0.001). A multivariate Cox proportional hazard analysis, performed on the entire group, identified unfavorable cytogenetic risk group, advanced age (> 60), FLT3-ITD and AML-MRC status as significant predictors of worse OS with the following respective hazard ratios: 2.82 (95% CI, 1.52–5.26), 2.11 (1.01–4.42), 1.98 (1.01–3.90), 1.92 (1.01–3.65).Conclusion: The newly defined WHO category of AML-MRC exhibits a significantly worse clinical outcome compared to AML-NOS and is predictive of worse overall survival in the multivariate analysis of AML patients, independent of age or cytogenetic risk group. These findings support the clinical, morphologic and cytogenetic criteria for this 2008 WHO AML category. [Display omitted]

High Serum Level of Placental Growth Factor (PlGF) Predicts Adverse Overall Survival in Patients with Acute Myeloid Leukemia Treated with Standard Chemotherapy

Background: Angiogenesis plays an important role in the pathogenesis of acute myeloid leukemia (AML) and vascular endothelial growth factor (VEGF) is an essential positive regulator of this process. Placental growth factor (PlGF) is a member of VEGF family that exerts its angiogenic action by synergizing with VEGF. There is also evidence that PlGF may be survival factor for angiogenic endothelial cells. The role of PlGF in patients with acute leukemia have not been investigated at all.Material and methods: We measured the plasma concentrations of VEGF and PlGF in 61 patients with newly diagnosed AML and 22 healthy controls using the ELISA assay. Additionally, in AML patients we correlated the plasma levels of angiogenic cytokines with the number of viable (CEC) and apoptotic (CECAnnV+) circulating endothelial cells, known prognostic factors, response to induction therapy as well as overall survival (OS). The AML group consisted of 34 women and 27 men with a median age of 49 years (range 25–76 years). All patient received standard induction chemotherapy (according to 3+7 protocol). Statistical analysis included the following variables: VEGF and PlGF levels, age, sex, performance status, cytogenetic risk group according SWOG, presence of multilineage dysplasia, WBC and PLT count, hemoglobin level, LDH activity, % of bone marrow blasts and CEC as well as CECAnnV+ number.Results: The PlGF and VEGF median levels (23,6 pg/ml and 45,1 pg/ml respectively) were significantly higher in the AML at diagnosis than in the healthy subjects (9,5 pg/ml; p<0,0001 and 25,2 pg/ml; p<0,03 respectively). Moreover, the VEGF level at diagnosis in patients who did not respond to induction chemotherapy was significantly higher than in patients who achieved complete remission (CR) (84,7 vs 31,2 pg/ml; p<0,01). The significant negative correlation between the VEGF and percentage of apopototic CECAnnV+ was also observed.Median follow-up was 12,1 months (range: 1– 43 months). 50 patients (82%) achieved CR. The CR rate in low and high VEGF expressors was respectively 86% and 70% (p=0.07). The CR rate in high- and low PlGF expressors did not differ significantly (74% and 80% respectively). In univariate analysis poor cytogenetics was the only factor associated with increased risk of treatment failure. The median time of OS in analysed group was 15 months. In the univariate analysis high (over median) PlGF levels was associated with shorter OS (p=0.005) (Figure1).Shorter OS was also associated with age>40 years (p=0.009), failure to achieve CR (p=0.009) and higher (>50%) bone marrow infiltration with leukemic blasts (p=0.005). In the multivariate Cox proportional hazard model the elevated PlGF plasma level was strongly associated with shorter OS (p<0.007). The other factors found to influence shorter OS were: high bone marrow infiltration with leukemic blasts (p=0.02), age>40 years (p=0.04), failure to achieve CR (p=0.04).Conclusions: In conclusion we can state that the high plasma concentration of PlGF is associated with poor prognosis in AML patients. Observed in our study significantly higher PlGF level in AML patients compared to healthy controls may indicate that this angiogenic cytokine plays an important, independent of VEGF role in pathogenesis of AML [Display omitted]

The Genotype Consisting of Complex Karyotype and TP53 Gene Mutation Is Specific to Acute Myeloid Leukemia with Multilineage Dysplasia

Acute myeloid leukemia (AML) is a heterogeneous disorder of hematopoietic progenitor cells. The World Health Organization (WHO) classification of the myeloid neoplasm incorporates genetic, immunophenotypic, biologic and clinical features. Although the WHO classification recognizes several chromosomal translocations they are frequently seen in AML patients, for categorizing the AML with recurrent genetic abnormalities, a number of other genetic alterations such as point mutations and gene rearrangements have not been included. To date, several genetic alterations, which are involved in the pathogenesis of AML and associated with prognosis of patients, have been documented. Therefore, it is required to establish the detailed classification of AML according to the genetic status. In this study, we comprehensively analyzed the genetic alterations and in de nove AML patients and investigated the association with mutational status, cytogenetic status and clinical features in comparison with the WHO classification. The study population included 144 newly diagnosed de nove AML patients consisting of 38 recurrent genetic abnormalities (RGA), 34 multilineage dysplasia (MLD) and 72 not otherwise categorized (NOC) according to WHO subcategories. Bone marrow samples were obtained from the patients after obtaining informed consent for banking and molecular analyses. Mutations in FLT3, cKIT, NPM1, N-RAS, TP53, C/EBPA, AML1, WT1 and MLL/PTD were analyzed as previously described. In consistent with previous reports, FLT3 (24%), cKIT (5%), NPM1 (20%), N-RAS (8%), C/EBPA (12%), AML1 (2%), MLL-PTD (9%), WT1 (3%) and TP53 (8%) mutations were frequently observed. No significantly different distribution was found in the prevalence of FLT3, N-RAS, C/EBPA, AML1 and MLL-PTD mutations among the WHO categories. However, the skewed prevalence was found in cKIT, NPM1 and TP53 mutations: cKIT mutation was frequently found in AML-RGA, NPM1 mutation was not found in AML-RGA, and TP53 mutation was preferentially found in AML-MLD. N-RAS, C/EBPA, AML1 and WT1 mutations were not identified in AML-RGA, though their distributions were not statistically significant among the WHO categories. It is notable that 9 of 12 (75%) patients who have TP53 mutation are categorized in AML with MLD and 8 of the 9 have complex-karyotype. Importantly, 8 of 9 (88.9%) showed complex karyotype, and 5 of them deleted chromosome 17. In this study, we found 2 additional patients harboring TP53 mutation in the other categories, while they did not show the complex karyotype and chromosome 17 abnormality. Furthermore, complex karyotype was found in 14 of the 144 entire AML patients, while 10 of them were categorized in AML-MLD. The remaining 4 patients were categorized in AML-NOC. Three of them showed del(17) or del(17p), though TP53 mutation was not identified. The genotype consisting of complex karyotype and TP53 mutation was, therefore, specifically found in AML-MLD. Thus TP53 mutation and complex-karyotype AML are significantly correlated and associated with the presence of multi-lineage dysplasia (P= .000 and P= .000, respectively). Moreover, these patients have significantly inferior induction rate and overall survival in AML with MLD. These results suggest that TP53 mutation and complex-karyotype AML distinguish a disease entity in AML with MLD.

Aberrant immunophenotype in acute myeloid leukemia (AML): High frequency and association with dysplasia

17504 Background: Asynchronous expression of immunophenotypic markers on AML myeloblasts has been well described, but the association of aberrant phenotype with morphologic subclasses has not been reported previously. Methods: Multiparameter flow cytometry (MFC) data were analyzed for all patients (pts) diagnosed with AML at our institution from 2000–2006. MFC was done on fresh bone marrow aspirate and/or peripheral blood samples using the following panel of monoclonal antibodies in triple staining: CD2, CD3, CD4, CD5, CD7, CD8, CD10, CD11c, CD13, CD14, CD15, CD16, CD19, CD20, CD22, CD24, CD33, CD56, CD34, CD117, HLA-DR and TdT. Identification of myeloblasts was performed using Side Scatter (SS) characteristics and CD45 expression. Acute lymphoblastic leukemia (ALL) cases or cases with mixed lineage acute leukemia were excluded from the study. Results: We identified 76 pts with AML; median age 57, (range 10 weeks to 89 years), with 32 males and 44 females. The patients had the following French American British classification of AML: M0 (n=1), M1 (n=11), M2 (m=10), M3 (n=5), M4 (n=13), M5 (n=14), M6 (n=1), M7 (n=4) and not otherwise categorized non- M3 AML (n=17). Twenty two pts had AML with multilineage dysplasia (AML-MD) as classified by World Health Organization. Coexpression of TdT on myeloblasts was observed in 4 pts, one of these coexpressed CD5. T-cell lymphoid associated antigens (CD2, CD5 and/or CD7) were coexpressed on myeloblasts from12 pts. B cell lymphoid associated antigens (CD19 and/or CD20) were expressed on myeloblasts from11 pts. Both T and B cell lymphoid associated antigens were present on myeloblasts in 4 pts. Myeloblasts from 13 pts demonstrated the presence of the NK cell marker CD56. A total of 43 patients had AML with immunophenotypic lineage infidelity manifested by coexpression of one or more aberrant antigens on myeloblasts. The distribution of cases with lineage infidelity was higher in 22 pts with AML-MD (n=17, 77%) versus in 54 pts with no dysplasia (n=26, 48%), Chi square =5.4, p=0.02. Conclusions: Abnormal coexpression of lymphoid and NK cell markers was observed in significant number of AML pts and correlated with the presence of multilineage dysplasia. This finding may indicate an earlier stem cell origin of AML-MD. No significant financial relationships to disclose.

Overexpression of CXCR4 predicts adverse overall and event‐free survival in patients with unmutated FLT3 acute myeloid leukemia with normal karyotype

CXC chemokine receptor 4 (CXCR4) expression in acute myeloid leukemia (AML) is reported to correlate with FLT3 gene mutation and poorer prognosis. The prognostic significance of CXCR4 expression in patients with AML that have a normal karyotype and no evidence of FLT3 gene mutations was examined. The prognostic significance of CXCR4 expression in 122 AML patients with normal karyotype and no evidence of FLT3 gene mutation treated at our institution between 1997 and 2003 was analyzed. All patients received intensive chemotherapy according to institutional protocols; 84% received cytarabine-containing regimens. Bone marrow biopsy or clot specimens obtained before treatment were immunostained for CXCR4. There were 70 men and 52 women with a median age of 62 years (range, 22-82 years). Median follow-up was 18 months (range, <1-97 months). Seventy-six patients achieved complete remission (CR); 39 had recurrence. Sixty-six patients died, including 9 with no evidence of disease. CXCR4 was positive in 70 and negative in 52 patients, with CR rates of 58% and 71%, respectively (P = .09). Multivariate analysis demonstrated that CXCR4 expression, presence of multilineage dysplasia, and high creatinine level predicted poorer overall (OS) and event-free (EFS) survival. CONCLUSIONS.: The results suggest that CXCR4 expression is associated with poor prognosis in AML patients with an unmutated FLT3 gene.

Flexible Low-Intensity Combination Chemotherapy for Elderly Patients with Acute Myeloid Leukaemia

To evaluate the efficacy of a flexible low-intensity combination chemotherapy (FLICC) protocol in a multicentre, phase II study of elderly patients with acute myeloid leukaemia (AML). Twenty-five patients aged 61-78 years (median 70 years) with de novo (n = 17) or secondary (n = 8) AML (cytogenetic risk: favourable 2, intermediate 18, adverse 2, unknown 3) from eight Australian centres were enrolled. Treatment comprised mitoxantrone 6 mg/m(2) intravenously daily for 3 days, cytarabine 10mg/m(2) subcutaneously every 12 hours for 7-14 days and etoposide 100mg orally for 7-14 days. The treatment was generally well tolerated, and 13 patients (52%) achieved a complete remission (CR). One patient achieved a partial remission but died on day 28 due to pneumonia. Five patients (20%) had no response, whilst six (24%) died on or before day 30 and so were not evaluable. The median overall survival (OS) was 6.5 months, and the median remission duration was 7.7 months. Estimated 1-year survival was 32%, but patients achieving CR had an estimated 1-year survival of 64%, whereas none in the non-CR group survived to 1 year. Two of the CR patients have survived beyond 2 years. OS was significantly shorter in the adverse cytogenetic risk group of patients compared with the favourable- and intermediate-risk groups, with the rates of death relative to the adverse group being 0.02 and 0.08 in the favourable- and intermediate-risk groups, respectively. There was no significant association between CR rate and pre-existing myelodysplasia or the presence of multilineage dysplasia. The median durations of significant neutropenia (<0.5 x 10(9)/L) and thrombocytopenia (<20 x10(9)/L) with the first course of treatment in the 19 evaluable patients were 19 days (range 12-26) and 11 days (range 1-25), respectively. The median duration of stay in the hospital was 27 days (range 14-42). These values were much shorter for the second course of treatment: 6 days, 5 days and 15 days, respectively. The findings of this multicentre, phase II study validate the previously reported single-institution experience with the FLICC protocol in elderly patients with AML. The clinical outcome with this protocol is comparable to those reported with more aggressive anti-leukaemia protocols.