Higher Blast Percentage Research Articles

Gene Mutations and Minimal Residual Disease (MRD) As Predictors of Remission Duration in Adults with Core Binding Factor (CBF) Acute Myeloid Leukemia (AML) Treated with High-Dose Cytarabine (HDAC) - First Results of the Prospective French Intergroup CBF-2006 Trial

Abstract 410 Background.CBF-AML is a favorable AML subset cytogenetically defined by t(8;21) or inv(16)/t(16;16) rearrangements, respectively responsible for RUNX1-RUNX1T1 (CBFa) or CBFB-MYH11 (CBFb) gene fusion. Nonetheless, relapse incidence may reach 30–40% in these patients. Even if not yet prospectively validated, early MRD monitoring based on chimeric fusion gene transcript quantification by RQ-PCR may help to define patients at higher risk of relapse. Detection of activating KIT and FLT3 gene mutations has also been retrospectively associated with higher relapse rate in these patients. The French CBF-2006 trial (NCT00428558) aimed to prospectively evaluate both markers as predictors of remission duration in homogeneously treated patients. Methods.Patients (18–60y) with newly diagnosed CBF-AML were eligible. All were studied for KIT exon 8/17, FLT3-ITD, FLT3-D835, N-RAS and K-RAS mutations. They were randomized to receive timed-sequential or standard induction. Timed-sequential induction was cytarabine (AraC) 500 mg/m2 CI D1-3 and daunorubicine (DNR) 60 mg/m2 D1-3, followed by AraC 1 g/m2/12h D8-10 and DNR 35 mg/m2 D8-9. Standard arm was AraC 200 mg/m2 CI D1-7 and DNR 60 mg/m2 D1-3, followed by the second sequence at D16-18 only in case of persistent bone marrow (BM) blasts at D15. Patients in first complete remission (CR1) received 3 HDAC consolidation cycles with AraC 3 g/m2/12h D1/3/5. Fusion gene expression was quantified at baseline and before each HDAC cycle (MRD1, MRD2, MRD3) by the 100 × fusion gene/cABL ratio. Patients not reaching a 3-Log ratio reduction at MRD2 were offered allogeneic stem cell transplantation (SCT) in CR1 if they had an HLA-identical donor or could enter a single-agent dasatinib sub-study (see Boissel et al., this meeting). Results.Among the planned 200 patients randomized, 198 met eligibility criteria (96 CBFa and 102 CBFb, compared in Table 1). Overall, incidences of KIT, RAS, FLT3-D835 and FLT3-ITD mutations were 21%, 29%, 10% and 6%, respectively. Patients with KIT and FLT3-D835 mutations had higher WBC (P=0.04 and 0.02) and BM blast percentage (P=0.0004 and 0.05). Those with RAS mutations had lower BM blast percentage (P=0.01) and baseline transcript ratio (P=0.001). Overall, 196 patients reached CR1 and 55 relapsed. At 3 years, probabilities of durable remission, disease-free (DFS) and overall survival from CR were 64%, 61% and 84%, respectively, without differences between the CBF subsets or induction arms. Fifty-five patients did not reach the 3-Log MRD2 reduction (18 CBFa and 37 CBFb, P=0.006), 9 receiving SCT in CR1. Poor MRD2 response correlated with age (P=0.01), WBC (P=0.04), KIT mutations (P=0.04), and initial transcript ratio (P=0.004). Among the characteristics listed in Table 1, shorter remission duration was associated with high WBC (P=0.011), high BM blast percentage (P=0.062), del(9q) (P=0.041), KIT mutation (P=0.048), FLT3-D835 mutation (P=0.077), high initial transcript ratio (P=0.053), and <3-Log MRD2 reduction (P=0.006) in univariate analysis stratified by CBF subtype and treatment arm. In multivariate analysis, the three latter factors, but not KIT mutations, remained independently predictive of shorter remission duration and DFS (P= 0.014, 0.033, 0.002 and 0.001, 0.012, 0.005, respectively). Censoring patients receiving SCT in CR1 yielded similar results. Conclusions.This prospective study of 198 CBF-AML patients homogeneously treated with HDAC revealed significant correlations between initial characteristics and response to therapy. Prognostic analysis did not find that KIT mutations had independent impact on outcome, which was rather influenced by baseline fusion transcript ratio and post-consolidation MRD reduction (reminiscent of Schnittger et al. 2003), and FLT3-D835 mutation. Ongoing pan-genomic analyses might help to further dissect CBF-AML heterogeneity.Table 1Patient characteristicsCBFa (N = 96)CBFb (N = 102)P valuesMedian age42y42y0.37Median WBC, G/L1122<0.001Median BM blast percentage52570.10Therapy-related AML1020.016Loss of Y chromosome*330<0.001Tri 22*018<0.001Tri 8*2150.002Del(9q)*141<0.001Del(7q)/–7*450.99KIT exon 8/17/both mutation**5/16/112/6/00.38FLT3 D835/ITD/both mutation**4/6/015/4/10.11N/K-RAS mutation**13/228/110.001Median baseline transcript ratio30389<0.001>3-Log ratio reduction at MRD2***71580.006*196,**194, and***184 patients studied. Disclosures:No relevant conflicts of interest to declare.

Expression of topoisomerase (topo) II in adult acute myeloid leukemia (AML): Relationships to immunophenotype and treatment outcomes.

6564 Background: Anthracyclines used in the treatment of AML target topo II. Topo II α and β are genetically distinct with different expression (expr) patterns and cellular distribution. While topo IIα has been associated with increased proliferation and an adverse prognosis in AML, there is no information regarding topo IIβ. This study examined the relationships of topo IIα and β expr to immunophenotype (IP), multidrug resistant (MDR) phenotype and outcomes in adult AML. Methods: 230 pts with non-M3 AML by FAB criteria who received frontline ara‐C/daunomycin (AD) therapy during the years 1992-98 on studies SWOG-9031, ‐9126, ‐9333 or ‐9500 were included (median age 63 years [18-88], WBC 29x109/L [0.8-274]). RT-PCR of RNA from marrow blasts was run with topo IIα and β specific primers (ABI PRISM 7900HT). Topo IIα and β expr in pts, normalized to internal standard β2‐microglobulin, was measured by log (fold-change) relative to the Kasumi AML cell line. Treatment outcomes - complete response (CR), resistant disease (RD), and relapse-free and overall survival (RFS, OS) - were analyzed by logistic or Cox regression. Results: Topo IIα and β data were available for 211 pts. Topo IIα expr in pts ranged from 298-fold less to 91-fold more than in the Kasumi AML cell line, but was not significantly correlated with any pt characteristic, IP or treatment outcome in uni- or multivariate analysis. In contrast, topo IIβ ranged from only 9‐fold less to 10-fold more than the cell line, but was associated with higher blast percentage in marrow (P=0.0001) or blood (P=0.0055); higher CD7 (P=0.0071); and lower CD14 (P<0.0001) and CD54 (P<0.0001) in multivariate regression analysis. In 105 pts with MDR data, topo IIβ was also inversely correlated with Rhodamine efflux (P<0.0001) and CD11c (P=0.0010). However after adjusting for prognostic factors, topo IIβ was not significantly related to any outcome, nor did topo IIβ interact significantly with any prognostic factors. Conclusions: Topo IIβ expr reflects aspects of disease biology, such as highly proliferative disease (higher blasts), IP and MDR, and thus may not be an independent variable influencing outcome in adult AML pts treated with anthracycline-based therapy.

Negative Effects of GM-CSF Signaling In t(8;21)-Induced Leukemia

AbstractAbstract 3163The t(8;21)(q22;q22) translocation is one of the most common chromosomal translocations in de novo acute myeloid leukemia (AML). The 8;21 translocation is often associated with additional cytogenetic abnormalities. The loss of the sex chromosome (LOS) is by far the most frequent abnormality found in association with the t(8;21) leukemia, accounting for 32–59% of patients, in contrast to other types of AML in which the LOS occurs in less than 5% of patients. To evaluate the role of sex chromosome deletion in t(8;21)-related leukemogenesis, hematopoietic cells from a mouse line with only one sex chromosome were used in retrovirus-mediated t(8;21) (AML1-ETO) expression and transplantation assays. The absence of leukemia in those animals suggested that a gene present in the pseudoautosomal region of sex chromosomes in humans but not in mice may be the target gene in LOS. The granulocyte-macrophage colony-stimulating factor receptor α (GM-CSFRα) gene is one such gene and is also known to be involved in myeloid cell survival, proliferation and differentiation. The GM-CSFRα gene is specifically down-regulated in AML patients with t(8;21), but not in other common translocations (Valk PJM et al, NEJM, 2004). The GM-CSFR complex is composed of α and βc subunits that assemble into a complex for receptor activation and signaling. To investigate the role of GM-CSFR signaling in t(8;21)-mediated leukemogenesis, GM-CSFR common β subunit knockout (GM-CSFRβc-/-) mice were used in our studies as a model for deficient GM-CSFR signaling. Transduction of AML1-ETO in hematopoietic cells from GM-CSFRβc-/- resulted in myeloid leukemia of a median survival time of 225 days, high percentage of blasts in peripheral blood and bone marrow, anemia, thrombocytopenia, hepatomegaly and splenomegaly. Comparison of wild-type and GM-CSFRβc-/- cells in the same transplantation resulted in development of AML1-ETO-induced leukemia at higher penetrance in GM-CSFRβc-/- cells (28.5% vs 100%). Moreover, the latency of leukemia was shorter in GM-CSFRβc-/- cells than in wild-type cells after transduction of AML1-ETO9a. Analysis of the hematopoietic compartment of healthy GM-CSFRβc-/- mice detected no significant abnormalities in the immature hematopoietic compartment (LSK, CMP, GMP, MEP), suggesting that AML1-ETO expression is required for leukemia to occur. In vitro, expression of AML1-ETO alone is sufficient for the immortalization of normal hematopoietic cells, as demonstrated by serial replating capacity of cells in methylcellulose colony assay. Addition of mGM-CSF to the basic cytokine cocktail (mIL-3, hIL-6, mSCF, hEPO) did not significantly affect number, type, size, and cell composition of colony cells. In contrast, the addition of mGM-CSF eliminated the replating capacity of AML1-ETO expressing cells, although they survived longer than control vector-infected cells. The results suggest that activation of GM-CSF signaling can specifically abrogate the self-renewal ability of potential leukemic stem cells in the early immortalization phase. These results support a possible tumor suppressor role of GM-CSF in leukemogeneis by AML1-ETO and may provide clues to understand how AML1-ETO corrupts normal GM-CSF signals to its own advantage for leukemogenic transformation. Disclosures:No relevant conflicts of interest to declare.

The Proportion of Ph+ CD34+CD38neg Leukemic Stem Cells In the Bone Marrow of Newly Diagnosed Patients with Chronic Myeloid Leukemia (CML) In Chronic Phase (CP) Is Variable and Correlates with High Sokal Risk, High Leukocyte Count, Low Hemoglobin Concentration, Splenomegaly and Increased Hematological Toxicity During Initial TKI-Therapy. Data From a Randomized Phase II NordCML006 Study

AbstractAbstract 667 Background:Targeted tyrosine kinase inhibitor (TKI) therapy efficiently induces rapid hematologic and cytogenetic responses in most CML patients. In vitro studies have suggested that CML stem cells are resistant to TKIs and therefore the treatment may need to be life-long. However, the in vivo effects of TKIs on the leukemic stem cell pool in a patient population have not been prospectively assessed. In addition, the biological impact and prognostic value of leukemic stem cell burden at diagnosis is unknown. Aim:To analyze the proportions of Ph+ cells in the stem cell compartment in newly diagnosed CP CML patients at diagnosis, and correlate the initial leukemic stem cell burden to biological variables and hematological toxicity during first 3 months of TKI therapy. Patients and Methods:42 newly diagnosed CP CML patients within the Nordic countries were randomized to receive either dasatinib 100 mg (n=21) or imatinib 400 mg (n=21) once daily. Stem cell assays were performed at diagnosis and at 1, 3, and 6 months from start of TKI therapy. After pre-selection of CD34+ cells from large volume bone marrow (BM) aspirates with paramagnetic beads, the CD34+ cells were fractionated into CD38 positive and negative pools using a sorting flow cytometer. The proportion of Ph+ cells in the stem cell fractions was assayed by counting 1000 cells with interphase FISH for BCR-ABL1. Results:Measurement of Ph+ stem cells was feasible in most patients at diagnosis and results from 36 evaluable patients will be presented. The median volume of BM aspirate was 28, 37, 40 and 36 ml at diagnosis, 1, 3, and 6 months after therapy start, respectively. The median yield of BM mononuclear cells was 1000, 110, 93 and 79 ×106, respectively. The median proportion of Ph+ cells was significantly lower in the more primitive CD34+CD38neg fraction when compared to the CD34+CD38+ fraction or to unfractionated BM (79%, range 0.6–100%; 96%, 50–100%; and 96%, 57–100%, respectively, p=0.0001). The proportion of Ph+ cells in the CD34+CD38neg fraction at diagnosis correlated with high leukocyte count (r=0.59, p<0.001), enlarged spleen size (r=0.46, p=0.005), low hemoglobin concentration (r=-0.46, p=0.006) and high blast percentage in peripheral blood (r=0.62, p<0.001). No correlation was found to age, gender, platelets, eosinophils or basophils. The proportion of Ph+ cells in the CD34+CD38+ fraction correlated only to the leukocyte count (r=0.33, p=0.049). Patients with high Sokal risk had a significantly higher proportion of Ph+ CD34+CD38neg stem cells at diagnosis as compared to low and intermediate risk patients (94% vs. 75%, respectively, p=0.036). Patients who had a higher leukemic stem cell burden (more than the median value of 79% of Ph+ CD34+CD38neg cells, Table 1) at diagnosis experienced more grade ≥2 hematological toxicity (neutropenia in particular) during first 3 months of TKI therapy as compared to patients with a lower (<79%) stem cell burden (62% vs. 19%, respectively, p=0.027). [Display omitted] Conclusions:The proportion of Ph+ stem cells at the time of diagnosis varied from 1 to 100% between individual CML patients. It was correlated with hemoglobin concentration, leukocyte count, blast percentage and spleen size at diagnosis and with hematological toxicity during early course of treatment, mirroring paucity of healthy hematopoietic stem cell reservoir. The size of the leukemic stem cell pool at diagnosis may be a powerful prognostic marker and a major biological determinant for the high Sokal risk group. The effect of TKI therapy on the malignant stem cell pool size and correlation to therapy responses will be evaluated when all patients have reached the primary study endpoint of 6 months. Disclosures:Mustjoki:BMS, Novartis: Honoraria. Richter:BMS, Novartis: Consultancy, Honoraria. Simonsson:BMS, Novartis: Consultancy, Honoraria, Research Funding. Porkka:BMS, Novartis: Consultancy, Honoraria, Research Funding. Hjorth-Hansen:BMS, Novartis: Consultancy, Honoraria, Research Funding.

A Case of Therapy-related ALL with MLL Gene Rearrangement Following Treatment of Breast Cancer

ALL with MLL gene rearrangement secondary to chemotherapy has been rarely reported. We report a case of therapy-related ALL (t-ALL) with MLL gene rearrangement in a patient who had undergone treatment for breast cancer. A 60-yr-old woman with breast cancer underwent breast-conserving surgery followed by 6 cycles of adjuvant chemotherapy (cyclophosphamide, epirubicin, and fluorouracil) and radiation therapy (dose, 5,040 cGy to the left breast and a 1,000 cGy boost to the tumor bed). A follow-up examination performed 14 months after the chemotherapy revealed no evidence of breast malignancy. However, the patient's complete blood cell count indicated acute leukemia: white blood cell count, 174.1 x 10(9)/L with 88% blasts; Hb level, 12.5 g/dL; and platelet count, 103.0 x 10(9)/L. Examination of the bone marrow aspirate smear revealed a high percentage of blasts (85.1% of all nucleated cells); the blasts showed a pro-B immunophenotype and were positive for CD19, CD79a, HLA-DR, CD34, and terminal deoxynucleotidyl transferase (TdT). Cytogenetic and FISH analyses revealed t(4;11)(q21;q23) and MLL gene rearrangement, respectively. The patient received induction chemotherapy with cyclophosphamide, vincristine, doxorubicin, and dexamethasone and achieved complete remission. Following consolidation chemotherapy, she underwent allogenic peripheral blood stem cell transplantation and has been clinically stable. To our knowledge, this is the first reported case of t-ALL with MLL gene rearrangement following treatment of breast cancer in Korea.

Mutation and Single-Nucleotide Polymorphism (rs16754) in Wilms Tumor-1 Gene Are Independent Prognostic Factors in Acute Myeloid Leukemia.

Abstract 995Poster Board I-17Several reports have suggested that mutations in the Wilms tumor 1 gene (WT1) represent an adverse prognostic factor in acute myeloid leukemia (AML). Here we examined the associations of WT1 mutations in exons 7 and 9 and the silent R301 single-nucleotide polymorphism (SNP) in exon 7 (A903G; NCBI dbSNP reference ID: rs16754) with outcome in AML patients treated at a single institution. Peripheral blood plasma and bone marrow samples from 174 newly diagnosed AML patients were tested for WT1 mutations in exons 7 and 9 by sequencing and fragment-length analysis for the detection of small deletions/insertions. Sequencing provided information on the specific genotype of the rs16754 SNP. The findings were correlated with outcome and other laboratory findings. WT1 mutation was detected in 7 of 50 (14%) AML patients <50 years of age and in 5 of 124 (4%) patients >50. The silent R301 SNP was detected at frequencies of 4% for GG, 26% for GA, and 70% for AA genotypes, but there was no difference with age. A similar SNP genotype distribution was detected in normal control subjects. WT1 mutations were associated with higher white cell count (P=0.01) and higher percentage of blasts in bone marrow (P=0.03) and peripheral blood (P=0.009). In addition, WT1 mutation was significantly associated with FLT3 mutation (P=0.002) but not NPM1 mutation (P=0.8). WT1 mutation was also significantly associated with shorter survival (P=0.025), event-free survival (P=0.002), and complete remission duration (P=0.002) in patients <50 years of age, but not in older patients. The association with shorter survival persisted when only patients with intermediate cytogenetics were considered (P=0.03). There was no correlation between WT1 mutation and response to therapy. The most striking correlation was between the presence of the GG genotype at R301 and longer survival, irrespective of age (see survival chart below). There was no difference in survival between the AA and GA groups. To the best of our knowledge, this is the first report describing correlation between survival duration and an SNP in the WT1 gene. While we confirm that the presence of WT1 mutation is associated with poor outcome in young AML patients, our data suggest that there is no clinical value in testing patients older than 50 for WT1 mutation, but that there may be value in testing adult patients of all age groups for the SNP polymorphism at R301 [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Acute Monoblastic/Monocytic Leukemia and Chronic Myelomonocytic Leukemia Share Common Immunophenotypic Features but Differ in the Extend of Aberrantly Expressed Antigens and the Amount of Granulocytic Cells.

Abstract 3799 Poster Board III-735 Acute monoblastic/monocytic leukemia (AMoL) is grouped into acute myeloid leukemia and related neoplasms by the 2008 WHO classification while chronic myelomonocytic leukemia (CMML) in grouped into myelodysplastic/myeloproliferative neoplasms (MDS/MPN). However, both entities share common features and sometimes may be difficult to differentiate from each other. In particular, monopoietic cells can be easily identified by multiparameter flow cytometry (MFC) based on their CD45/side-scatter (SSC) signal. However, immunophenotypes of monoblasts and dysplastic monocytes largely overlap. Their differentiation from each other is better accomplished by cytomorphology (CM) and the use of non-specific esterase. We compared cytomorphologic, immunophenotypic and cytogenetic results between 26 patients with AMoL and 139 patients with CMML which were analyzed at diagnosis between 2005 and 2009 in order to identify shared and discriminative characteristics. In AMoL vs. CMML 65.4% vs. 73.4% were male, the median age was 65.4 vs. 72.5 years (range 19.3-82.0 vs. 21.9-87.9 years, p&lt;0.001), and the median WBC count was 38.0 G/L vs. 10.4 G/l (range 0.8-126.0 G/l vs. 0.9-92.0 G/l, p&lt;0.001). Cytogenetics differed in that a normal karyotype was found in 79.1% of CMML cases and in only 38.5% of AMoL cases (p&lt;0.0001). Per definition, a t(9;11) (19.2%) was found only in AMoL (p&lt;0.001) which was almost identical for other translocations involving MLL (15.4% vs. 0.7%, p=0.003). Other cytogenetic abnormalities did not significantly differ between AMoL and CMML. First, immunophenotypes of monopoietic cells were compared between AMoL and CMML. While an aberrant coexpression of CD56 was present in all cases with AMoL this was true in only 82.0% of CMML cases (p=0.015). No other aberrantly expressed antigen was found in AMoL while in CMML a coexpression of CD2 was found in 21.6% (p=0.005), a lack of CD13 expression in 10.8% (n.s.), and a lack of HLA-DR expression in 4.3% (n.s.). Next, we compared the frequencies of different cell types between AMoL and CMML as identified by CM and MFC. In this analysis, granulocytic cells by CM included cells of all maturation grades from promyelocyte to granulocyte, which corresponds to the population identified in MFC by their dim expression of CD45 and their strong SSC signal. As anticipated when considering the definition of the respective entities CM identified higher percentages of blasts (mean±SD 78.7±13.8 vs. 8.6±5.1, p&lt;0.001) and lower percentages of monocytes (1.7±3.9 vs. 13.8±11.9, p&lt;0.001) and granulocytic cells (7.9±7.4 vs. 53.8±15.8, p&lt;0.001) in AMoL vs. CMML. Applying the blast gate in the CD45/side-scatter analysis for the identification of blasts (CD45(+)SSC-) MFC also found higher percentages of blasts in AMoL vs. CMML (18.2±25.1 vs. 4.0±3.2, p&lt;0.001) and monopoietic cells were found also at higher frequencies in AMoL (28.6±27.4 vs. 20.1±12.3, p=0.012). Interestingly, however, MFC identified significantly higher frequencies of granulocytic cells in CMML (52.2±19.3 vs. 26.2±22.6, p&lt;0.001). Thus, while the CD45-SSC analysis is not capable of differentiating between monocytes and monoblasts it may be used to identify high percentages of granulocytic cells which is characteristic for CMML but not for AMoL. Based on these findings, the ratio M:G (monopoietic cells:granulocytic cells) was calculated for each case. The ratio M:G was significantly higher in AMoL as compared to CMML (mean±SD 5.2±10.4 vs. 0.8±2.3, p&lt;0.001). In conclusion, the present data demonstrate that AMoL and CMML after being discriminated according to WHO guidelines for cytomorphology share common features as identified by MFC but differ in the extend of aberrantly expressed antigens and the amount of granulocytic cells. For further studies and to support CM these parameters should be evaluated when using MFC as a diagnostic tool for AMoL and CMML. Disclosures: Kern: MLL Munich Leukemia Laboratory: Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership.

Fms-like Tyrosine Kinase (FLT) 3 and FLT3 Internal Tandem Duplication in Different Types of Adult Leukemia: Analysis of 147 Patients

To assess the expression level of fms-like tyrosine kinase 3 (FLT3), the incidence of FLT3/internal tandem duplications (ITD) mutation, and prognostic value of FLT3 changes in different types of adult leukemia. Bone marrow mononuclear cells were isolated from 147 adult patients with leukemia. Reverse transcriptase polymerase chain reaction (PCR) was used to screen FLT3/ITD mutation and quantitative PCR was performed to evaluate the expression of the FLT3 transcript. Flow cytometry was used for detection of FLT3 receptor protein expression on bone marrow mononuclear cells. Pearson correlation analysis was performed to estimate the significance of FLT3. FLT3 expression was higher in acute myeloid leukemia and B-acute lymphoid leukemia than in T-acute lymphoid leukemia (P=0.006, P=0.001) and chronic myelogenous leukemia (P<0.001). In chronic myelogenous leukemia, FLT3 expression in blast transformation phase was higher than in acceleration phase (P=0.023). Surface expression of FLT3 protein was correlated with high percentage of bone marrow blasts and with FLT3 mRNA expression (r=0.366, P<0.001) in acute leukemia. FLT3/ITDs in the juxtamembrane domain were found in 25% of patients with acute myeloid leukemia and 7% of patients with acute lymphoid leukemia. FLT3/ITD positive sequences had 36, 42, and 57 nucleotides. FLT3/ITD mutation was associated with a higher white blood cell count, higher marrow blast percentage, and elevated serum lactate dehydrogenase (P=0.045, P=0.014, P<0.001, respectively) and not associated with a higher FLT3 mRNA and FLT3 protein expression, and lower complete remission (P=0.091, P=0.060, P=0.270, respectively). FLT3 expression levels differed in different types of adult leukemia. Overexpression of FLT3 and presence of a positive FLT3/ITD mutation in acute leukemia were associated with unfavorable clinical characteristics and poor prognosis.

Expression of myeloperoxidase enhances the chemosensitivity of leukemia cells through the generation of reactive oxygen species and the nitration of protein

Myeloperoxidase (MPO), a pivotal lineage marker for acute myeloid leukemia (AML), has been also shown to have a prognostic value: a high percentage of MPO-positive blasts correlates to favorable prognosis. To understand the relationship between the expression of MPO in leukemia cells and the response to chemotherapeutic agents, we established MPO-expressing K562 leukemia cell lines and then treated them with cytosine arabinocide (AraC). Cells expressing wild-type MPO, but not mutant MPO that could not mature, died earlier of apoptosis than control K562 cells. Reactive oxygen species (ROS) were generated more in leukemia cells expressing MPO, and the generation was abrogated by MPO inhibitors or antioxidants. Tyrosine nitration of cellular protein also increased more in MPO-expressing K562 cells than control cells after treatment with AraC. In clinical samples, CD34-positive AML cells from high-MPO cases showed a tendency to be sensitive to AraC in the colony-formation assay, and the generation of ROS and the nitration of protein were observed only when the percentage of MPO-expressing cells was high. These data suggest that MPO enhances the chemosensitivity of AML through the generation of ROS and the nitration of proteins.

Marrow fibrosis predicts early fatal marrow failure in patients with myelodysplastic syndromes

Marrow fibrosis (MF) has rarely been studied in myelodysplastic syndromes (MDS). There are no data on occurrence and significance of MF in the context of the World Health Organization (WHO) classification of disease. In total, 349 bone marrow biopsies from 200 patients with primary MDS were examined for MF and its prognostic relevance. MF correlated with multilineage dysplasia, more severe thrombopenia, higher probability of a clonal karyotype abnormality, and higher percentages of blasts in the peripheral blood (P<0.002). Its frequency varied markedly between different MDS types ranging from 0 (RARS) to 16% (RCMD, RAEB, P<0.007). Two patients with MF showed a Janus kinase-2 mutation (V617F). Patients with MF suffered from marrow failure significantly earlier with shortening of the survival time down to 0.5 (RAEB-1/-2), and 1-2 (RCMD, RA) years in median (P<0.00005). The prognostic relevance of MF was independent of the International Prognostic Scoring System and the classification of disease. The risk of MF Differs markedly between various subtypes of MDS. MF indicates an aggressive course with a significantly faster progression to fatal marrow failure and should therefore be considered in diagnosis, prognosis and treatment of disease.

G-CSFSR Mutations Present in Patients with Severe Congenital Neutropenia Cooperate with PML-RARα To Induce Acute Myeloid Leukemia in Mice.

Severe congenital neutropenia (SCN) is an inherited disorder of granulopoiesis that is associated with a markedly increased risk of developing acute myeloid leukemia (AML) or myelodysplasia (MDS). Somatic mutations of CSF3R, encoding the G-CSF receptor (G-CSFR), are strongly associated with the development of AML/MDS in SCN. These mutations invariably produce a truncated G-CSFR that, though remaining ligand-dependent, transmits a hyperproliferative signal. Transgenic mice carrying a targeted (knock-in) mutation of Csf3r (termed d715) reproducing a mutation found in a patient with SCN have an exaggerated neutrophil response to G-CSF treatment but do not develop AML/MDS. Moreover, we recently showed expression of the d715 G-CSFR confers a strong clonal advantage at the hematopoietic stem cell level that is dependent upon exogenous G-CSF. Collectively, these data suggest that CSF3R truncation mutations are an initiation or early progression factor for leukemic transformation. However, there is, as yet, scant direct evidence supporting this hypothesis. Previous studies have established that activating mutations of receptor tyrosine kinases, such as internal tandem duplications of FLT3, are able to cooperate with PML-RARα to induce AML. Since the CSF3R mutations in SCN also are “activating”, we asked whether the d715 G-CSFR could cooperate with PML-RARα to induce AML in mice. PML-RARα transgenic mice were intercrossed with d715 G-CSFR mice (all inbred > 10 generations onto a C57BL/6 background) to generate the cohorts listed in Table 1. A separate cohort for each genotype was treated chronically with pegylated G-CSF (1 mg/kg every 4–5 days for 6 months) to simulate the high level of serum G-CSF present in patients with SCN. Complete blood counts were performed at 3 months intervals and documented a similar increase in neutrophil counts in all mice treated with G-CSF. The cumulative incidence of AML and median follow-up for each cohort is shown in Table 1. None of the mice without the PML-RARα transgene, regardless of G-CSF treatment, developed AML, confirming that the d715 G-CSFR is not sufficient to induce AML. In mice carrying the PML-RARα transgene but not treated with G-CSF, a nonsignificant trend to increased AML was observed in mice expressing the d715 G-CSFR (P=0.12). However, in mice carrying the PML-RARα transgene and treated with G-CSF, the presence of the d715 G-CSFR significantly increased the penetrance (P=0.009) and reduced the latency of AML. In all cases, the leukemia was characterized by leukocytosis, splenomegaly, and a high percentage of blasts in the bone marrow and spleen that co-express Gr1 and c-Kit. These data provide the first direct evidence that the CSF3R mutations present in patients with SCN are leukemogenic and provide further support for the proposition that patients who acquire CSF3R mutations be considered for early stem cell transplantation.The cumulative incidence of AML and median follow-up for each cohortPML-RARCSF3RG-CSF RxNMedian follow-upCumulative AML%NoWTNo204540.0%Nod715No452500.0%NoWTYes204880.0%Nod715Yes473110.0%YesWTNo572866.4%Yesd715No6726711.9%YesWTYes5432220.4%Yesd715Yes5031144.0%

High Blast Percentage, Chromosome 17 Abnormalities and Severe Pancytopenia Predict Death within Twelve Months in Patients with Myelofibrosis.

Existing prognostic models for myelofibrosis (MF) are based on complete blood count parameters and clinical symptoms at the time of initial presentation, with the most widely cited being the Lille score (Blood 88.1013). These models, however, are only applicable to patients at the time of initial diagnosis, and they do not identify the patients at imminent risk of death. In order to identify patients in advanced phase (AP) MF that may be candidates for intensified therapy, we reviewed the records of 371 MF patients for disease features that predict for survival less than twelve months. Median baseline characteristics (range): age 63 yrs (24–86); male 59%; post-PV or post-ET MF 21%; spleen 5cm (0–30); HB <10g/dL or transfusion dependent (TD) 53%; abnormal cytogenetics 126/337 (37%). Patients presented at a median of 4 months (range, 0 to 296 months) from initial diagnosis; 6% were previously treated with alkylators or radiation, and 36% with hydroxyurea. Median follow-up was 46 months for survivors. Independent predictors of poor survival at initial presentation were: abnormal chromosome 17 (ABN17; p<0.001); age (p<0.001); hepatomegaly ≥5cm (p<0.001); platelets <100 (p<0.001); HB <10 or TD (p<0.001); abnormal WCC (p=0.007); PB or BM blasts ≥10% (p=0.008); splenomegaly ≥20cm (p=0.02); and male gender (p=0.02). Factors significant on univariate analysis but not significant on multivariate modelling included primary vs secondary MF, other cytogenetic abnormalities, previous radiation / alkylator or erythropoietin exposure, monocytes ≥1×10^9/L, abnormal creatinine or bilirubin, and performance status. Three disease features were chosen as criteria for AP because of their association with median survival <12 months: PB/BM blasts ≥10% (n=19, survival 10 months), ABN17 (n=9, survival 5 months) and severe cytopenia not related to therapy (PLT <25 and WCC <4.0, n=9, survival 8 months). These criteria were independent of existing prognostic models: among patients with a high-risk Lille score, median survival was significantly inferior for patients with AP criteria (n=19, 5 months) compared to those without (n=55, 24 months p<0.0001). In order to validate these criteria, we followed the progress of 334 patients not in AP at presentation. During follow-up, 56 additional patients met AP criteria, and median survival from AP development were: PB/BM blasts ≥10% (n=38), 12 months; ABN17 (n=6), 6 months; severe cytopenia not related to therapy (n=12), 10 months. The actuarial risk of AP development was 23% at 4 years. Of the 93 patients in AP, blast phase (BP) occurred in 30 (32%), and a further 46 (49%) died without reaching BP. In contrast, BP occurred in only 9 of 278 (3%) of patients in chronic phase (p<0.0001). Our observations indicate that PB/BM blasts ≥10%, ABN17 and severe cytopenia evolve during the natural history of primary or secondary MF and are associated with a high risk of blastic transformation or death from progressive disease. Recognition of such patients as AP is an important step in facilitating the development of high-risk intervention strategies.

Risk Factors for Disease Progression in del(5q) MDS Patients Treated with Lenalidomide.

Introduction: Lenalidomide is a novel immunomodulatory drug that is highly effective in patients with transfusion-dependent MDS with del(5q.31) chromosomal abnormality. In the recent MDS-003 study of lenalidomide, there was a 76% erythroid response rate, including 67% transfusion independence. Cytogenetic complexity or bone marrow blast percentage did not affect this response rate. A number of patients experienced disease progression to higher FAB subtypes or AML. We questioned whether lenalidomide might promote disease progression in del(5q) MDS and performed a retrospective analysis to identify risk profiles.Methods: Fifty patients from three institutions were included in this analysis. They were partly treated within the Lenalidomide-MDS003-study. Patients were treated with an initial lenalidomide dose of 10 mg po daily. In case of grade >2 neutropenia, G-CSF and antibiotics were administered.Results: The median age was 71 years; 31 patients were female and 20 were male. Disease progression to a higher FAB subtype or to AML occurred in 13 patients (29.5%). 7 of the 13 patients had RAEB at the first lenalidomide dose. In addition, 3 of these had additional chromosomal aberrations (2, trisomy 21; 1, complex karyotype). Of the remaining 6 patients, 2 had a complex karyotype at the first lenalidomide dose, 1 had an additional inv(9)(p11q12), and 1 had hypocellular bone marrow so no FAB subtype could be assigned. Only 2 of 50 patients (4.3%) with 5q-syndrome progressed to AML; both patients developed acute erythroid leukemia (FAB M6).Conclusion: Within the del(5q) MDS subgroup, patients with an isolated del(5q) chromosomal aberration and a bone marrow blast count of <5% have the longest overall survival. Patients with additional chromosomal abnormalities or a higher blast percentage have a much shorter overall survival and a higher risk for progressing to AML. Our data show that progression to higher MDS subtypes or AML occurs almost exclusively in patients with additional risk factors such as >5% bone marrow blasts or additional chromosomal anomalies. Lenalidomide does not seem to increase the risk of transition of del(5q) MDS to higher stages of disease.

Myeloperoxidase Increases Chemosensitivity of Leukemia Cells through the Generation of Reactive Oxigen and Nitrogen Species.

Myeloperoxidase (MPO) is a specific enzyme whose expression is restricted to the late stage of myeloid differentiation such as promyelocytes and more matured cells. In a pathological situation, however, morphologically immature blasts of acute myeloid leukemia (AML) express MPO at variety of percentages that has been utilized for the diagnosis of AML. On the other hand, the expression of MPO has been shown to have prognostic value for AML cases. Several groups including ours reported that the high percentage of MPO positive blasts at diagnosis was related to the favorable prognosis. Recently, we also found that the expression of the MPO gene in CD133 positive cells also related to the better prognosis of AML cases, suggesting the MPO in the very immature fraction, that is thought to contain leukemia stem cells, is related to the chemosensitivity of AML cells. These results made us hypothesize that MPO directly changes chemosensitivity of leukemia cells through its enzymatic activity. To test this hypothesis, we transfected wild type and enzymaticaly defective MPO (dMPO) cDNA into MPO-negative leukemia cells line, K562, generating K562-MPO and K562-dMPO. Control cells were K562-vector that were transfected with empty vector. The expression of MPO protein was shown in K562-MPO cells, but only precursor of MPO protein, apo-pro-MPO was expressed in K562-dMPO cells as expected. The enzymatic activity was shown only in K562-MPO cells. When these cells were treated with cytosine arabinoside (AraC), K562-MPO cells showed decreased proliferation by WST1 assay compared with wild type K562, K562-vector and K562-dMPO. The expression of Annexin V was increased in AraC-treated K562-MPO suggesting these cells died through apoptosis. Using flowcytometer or confocal microscopy with aminophenyl fluorescein (APF), reactive oxygen species (ROS) were detected in wild type K562 and K562-MPO cells after treatment with AraC. The amount of ROS shown by the intensity of APF-fluorescence increased more in K562-MPO compared with wild type K562 or K562-vector. The difference in the APF-fluorescence observed AraC-treated K562-vector and K562-MPO cells was strongly enhanced by the addition of hydrogen peroxide as a donor of ROS. However, this difference completely disappeared with the addition of N-acetyl cystein (NAC), an inhibitor of ROS, suggesting the involvement of MPO to generate ROS in K562-MPO cells. Although flowcytometer with DAF-2DA failed to detect the significant differences in the amount of reactive nitrogen species (RNS) among AraC-treated wild type K562, K562-vector and K562-MPO cells, immunoblotting against the nitrated tyrosine residue demonstrated the increased nitration of protein in K562-MPO cells compared with wild type or K562-vector. It was suggested that RNS was generated in K562-MPO treated with AraC. These results supported our working hypothesis that MPO in leukemia cells enhanced the sensitivity against chemotherapeutic agents through the generation of ROS and RNS.

Clinico-Biological Characteristics and Prognosis of Non-M3 Acute Myeloid Leukemia with High Percentage of Myeloperoxisase Positive Blasts. Single Center Experience.

Introduction: It has been suggested that acute myeloid leukemia (AML) showing mature phenotype is associated with favourable outcome. In a study recently published by JALSG, myeloperoxidase (MPO) positivity in over 50% of blasts had favourable prognostic impact, independent from karyotype, on achieving complete remission (CR), overall survival (OS) and disease free survival (DFS). No other studies have established the independent prognostic value of MPO expression.Objectives: Analyze the clinico-biological characteristics of AML with high percentage of MPO+ blasts and its impact on CR, OS and DFS.Material and methods: Between 1986 and 2005, 418 adult patients (median 53 years, range 15–80) were diagnosed with de novo non-APL AML and evaluated for percentage of MPO+ blasts. All patients received intensive chemotherapy. Diagnosis was made by optic microscopy of bone marrow (BM) aspirates stained with May-Grumwald giemsa, MPO, butyrate esterase and or non specific esterase. Cytogenetic and immunophenotype analysis was evaluated in 66% and in 76% of the cases respectively.Results: 118 patients (28%) showed a percentage of MPO+ blasts >75%. AML with MPO+ blasts >75% was associated with M1-M2-M4 subtypes, leucocytes >50×109/L, blasts in BM >70% and HLA-DR negativity (p<0.01). It was also significantly associated with favourable karyotype (11% vs 3% favourable, 52% vs 48% intermediate and 3% vs 15% unfavourable). Patients with AML and MPO+ blasts >75% obtained higher CR rate (71% vs 55%), due to less resistant disease (9% vs 22%, p<0.01). In multivariate analysis favourable karyotype, leukocytes <50×109/L and age <60 were favourable prognostic factors for CR. Median OS and DFS was higher in patients with AML and MPO+ blasts >75% (15 vs 7 months, p<0.001, y 41 vs 12 months, p<0.001, respectively). ). In multivariate analysis, favourable karyotype, leukocytes <50×109/L, age <60 years and MPO+ >50% were favourable prognostic factors for OS; and age <60 and MPO+ >75% were the only independent factors for DFS. Median DFS was higher in patients with AML and MPO+ blasts >75% in the intermediate cytogenetic risk group (59 vs 13 months, p=0.015), age <60 (109 vs 15 months, p=0.003), age >60 (13 vs 7 months, p=0.03), autologous stem cell transplantation (100 vs 9 months, p=0.04) and chemotherapy alone (16 vs 8 months, p=0.003).Conclusion: In our series, patients with AML and MPO+ blasts >75% show less chemoresistant disease and a longer remission duration, the latter independently from the karyotype. This biological characteristic could be useful in designing therapeutic strategies in patients that lack other prognostic markers.

Expression of LAT and PAG Adaptor Molecules in Blasts of Childhood Acute Lymphoblastic Leukaemias and in Normal B- and T-Precursors.

Despite the progress in leukaemia research, genetic background of some acute lymphoblastic leukaemias (ALL) is unknown. In the aetiopathogenesis of ALL the transmembrane adaptor proteins (including LAT and PAG) could play an important role as these proteins are key mediators of signalization cascades in lymphocytes.We analysed expression of PAG and LAT at the mRNA level by real-time quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR) and at the protein level by flow cytometry. In our study we assessed the expression levels in 10 patients with T-ALL and in 10 patients with B-cell precursor ALL (BCP-ALL). All analysed samples had a high percentage of malignant blasts (70–97%, median 91%). Sorted and unsorted bone marrow/peripheral blood samples of healthy volunteer donors were used as controls. To determine the PAG and LAT expression levels in normal immature thymocytes we analysed also sorted cells from the normal thymuses taken from children undergoing heart surgery. We used various differentiation markers of both B- (CD10, CD19, and CD20) and T-lineage (CD1a, CD3, CD4, CD7, CD8) for sorting to compare the leukaemic samples with the most adequate control in terms of maturation status of the cells. Informed consents were obtained from all volunteer donors or their guardians.Our data show that the expression of PAG at both protein and mRNA level during the physiological B-cell maturation is decreasing. On the contrary, PAG expression during the T-cell development and also the levels of LAT during the B- and T-cell maturation are increasing, however, the levels of LAT mRNA in B-precursors are very weak.In childhood BCP-ALL the expression of PAG on protein and mRNA level reflects the situation in corresponding non-malignant precursors with the exception of the TEL/AML1 positive leukaemias that show tendency to the overexpression of PAG. In the T lineage ALL we found a significantly lower expression of PAG and higher expression of LAT mRNA ((p<0.0001 and p=0.03, respectively, for mature T-ALL) compared to sorted thymocytes with corresponding immunophenotype. The LAT protein in BCP-ALL blasts is expressed very weakly or there is no expression at all. Also the levels of LAT mRNA are even lower than in the physiological BCP counterparts.We present the first study assessing changes in the expression of two important adaptor molecules at both mRNA and protein levels during the physiological maturation of lymphocyte precursors. Moreover, we demonstrate changes in the pattern of expression in childhood ALL and suggest that the aberrant transmission or processing of signal through the immunoreceptors of lymphoid precursors might play an important role in the aetiopathogenesis of the disease.

High Expression of CD40 on BCP-ALL Blasts Is an Independent Risk Factor Associated with Improved Survival and Enhanced Capacity To Upregulate the Death Receptor CD95.

Leukemogenesis is facilitated by deregulated survival signals and escape from immunosurveillance. For immunological control both adequate stimulation of effector cells as well as apoptosis sensitivity of target cells are required. The CD40 receptor, a member of the TNF/NGF receptor family, plays a pivotal role on normal B cells in both aspects as it upregulates the costimulatory molecules enhancing their antigen presenting capacity and the CD95 TNF receptor sensitizing cells to apoptotic stimuli. Due to its physiological immunomodulatory functions, we prospectively studied the prognostic impact of CD40 expression on primary precursor B (BCP)-ALL blasts of pediatric patients (pts) (n=121). Blasts exhibited an immunephenotype of c-ALL (n=81), preB-ALL (n=32) or proB-ALL (n=8). All pts (>1 and <17y) were treated according to the CoALL06-97 study protocol. The criteria for prognostic classification into high risk (HR; n=57) and low risk (LR; n=64) pts at initial diagnosis include immunephenotype, leukocyte count (WBC), age, genetic translocations and the in vitro resistance to chemotherapeutic agents (PVA score). In flow cytometry analysis blasts exhibited a broad range of CD40 expression with a mean of 80%±27% (±SD) positive blasts. In terms of the above mentioned prognostic discrimination criteria, we found significantly lower percentage of CD40 positive blasts in HR pts (median; range: 84%;3–100%) compared to the LR group (98%;3–100%) (p=0.007). With regard to prognostic relevance, pts with very high percentage (above the mean of 80%) CD40 positive blasts exhibited significantly better relapse free survival (RFS; 0.86±0.06 vs 0.65±0.09; p=0.009) in Kaplan Meier analysis and multivariate Cox regression analysis revealed that high CD40 expression was independently associated with superior RFS in our pediatric ALL pt cohort (p=0.006) in contrast to the risk factors age (p=0.72), WBC (p=0.33) and PVA score (p=0.60). In a subset of patients (n=34) we examined whether the level of CD40 expression corresponds to the capacity of blasts to upregulate costimulatory molecules and the CD95 death receptor upon CD40-ligation. CD40-crosslinking in samples with a very high percentage of CD40 positive blasts (>80%) and with intermediate/low expression of CD40 (<80%) did not result in significantly different upregulation of CD80 (37%;3–97% vs 41%;4–68%) and CD86 (83%;5–100% vs 72%;12–96%) in the two sample-sets (p=0.66 and p=0.86). In contrast, expression of CD95 in CD40-stimulated blasts showed a significant correlation with CD40 baseline expression (p=0.009) with a higher percentage of CD95 positive blasts following CD40-activation in samples containing >80% CD40 positive blasts compared to samples with intermediate/low CD40 expression (74%;17–95% vs 38%;9–94%; p=0.065). Thus, high percentage of CD40 positive blasts is an independent prognostic marker that identifies a group of pediatric BCP-ALL pts with favorable outcome. While the capacity to upregulate costimulatory molecules following CD40-activation might contribute to the observed improvement in survival, enhanced upregulation of the CD95 receptor seems to be the leading mechanism mediating sensitization towards apoptotic stimuli.

Having a higher blast percentage in circulation than bone marrow: clinical implications in myelodysplastic syndrome and acute lymphoid and myeloid leukemias

Determining the percentage of peripheral blood (PB) and bone marrow (BM) blasts is important for diagnosing and classifying acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS). Although most patients with acute leukemia or MDS have a higher percentage of BM blasts than PB blasts, the relative proportion is reversed in some patients. We explored the clinical relevance of this phenomenon in MDS (n = 446), AML (n = 1314), and acute lymphoblastic leukemia (ALL) (n = 385). Among patients with MDS or ALL, but not AML, having a higher blast percentage in PB than in BM was associated with significantly shorter survival. In multivariate analyses, these associations were independent of other relevant predictors, including cytogenetic status. Our findings suggest that MDS and ALL patients who have a higher percentage of PB blasts than BM blasts have more aggressive disease. These data also suggest that MDS classification schemes should take into account the percentage of blasts in PB differently from the percentage of blasts in BM.

The Combination of Intrinsic Sensitivity to Imatinib and Sokal Prognostic Score Is Strongly Predictive of Molecular Response in Newly Diagnosed CML Patients Treated with Imatinib.

The ability to predict which CML patients respond poorly to imatinib may facilitate a more aggressive approach to therapy in these patients. We have used western blotting to assess the phosphotyrosine content of the adaptor protein Crkl (p-Crkl) -a major substrate for BCR-ABL. PB was collected from 57 newly diagnosed CML patients prior to the initiation of therapy and dose dependent inhibition of Crkl phosphorylation, in the presence of imatinib, was measured following incubation of primary cells for 2 hours with varying doses of imatinib. Using this assay we were able to determine that the median IC50 for imatinib was 0.6 uM (R 0.375–1.8uM). For statistical analysis IC50's were then grouped into low and high relative to the median, and compared to molecular response achieved at both 3 and 12 months. Using Log Rank Survival Analysis there was a significant difference in the probability of achieving a 2 log depletion of BCR-ABL (measured by quantitative real-time Q-PCR) by 3 months between the two groups (Low IC50 30% n=31, High IC50 4% n=26; p=0.023 n=57). In addition the probability of achieving a 3 log reduction of BCR-ABL at 12 months (MMR, major molecular response) was significantly different (Low IC50 47%, High IC50 23%, p=0.034 ). We therefore assessed whether the IC50 was a surrogate measure of the Sokal Score or was an independent predictor of response. When we examined the median Sokal scores of both the low and high IC50 groups we found that there was no significant difference between the two groups (Low IC50 = 1.0, High IC50 = 0.9 p=>0.05 ) suggesting that IC50 was not a surrogate measure of Sokal. Log Rank survival analysis revealed that while Sokal Score did not predict for a 2 log depletion at 3 months (Low (43% n=32) vs Intermediate (21%n=25) vs High (21% n=29) p>0.05 ) it was predictive of a greater probability of MMR at 12 months (Low 53% vs Intermediate 42% vs High 21% p=0.044 ). The combined effect of these parameters was assessed:Probability of achieving a Major Molecular Response.AllLow SokalIntermediate SokalHigh SokalLow IC5047% n=3067% n=950% n=617% n=12High IC5023% n=2320% n=1022% n=90% n=4p-value0.0340.037>0.05>0.05Note: Not all Sokal Scores available at the time of analysis.Evidence presented here suggests that the IC50 level measured in blood cells from CML patients prior to imatinib therapy provides a statistically significant indicator of response at both early and late timepoints. Further to this, the combined measures of Sokal Score and IC50 provide a powerful tool for prediction of molecular response, which clearly delineates those patients groups who have a low probability of achieving a major molecular response. Preliminary investigations into the components of both Sokal and IC50 which may be contributing to these findings have highlighted a group of patients (n=7) who have a high blast percentage (5 to 21%) and low IC50. Unlike the majority of patients with low IC50 these patients all fail to achieve MMR. When these patients are removed from the low IC50 group the probability of MMR in the remaining patients is 71%. Further investigations into underlying mechansims for the inter-patient variation in IC50 are currently being undertaken.

High FLT3 mRNA Expression in Acute Myeloid Leukemia May Be Functionally an Alternative to Mutational Activation of the Receptor.

Mutations of the FLT3 gene are detectable in approximately 30% of all adult AML. These mutations lead to an autoactivation of the receptor inducing increased proliferation of the leukemic clone. An alternative mechanism of FLT3 activation might be mRNA overexpression. In the presented study the FLT3 expression level in 208 adult AML patients and 8 healthy donors was assessed by real time PCR and correlated to several parameters. In all patients cytomorphology, cytogenetics, and FLT3 mutation status was assessed. Significant differences of FLT3-expression levels were found in certain AML subgroups. The highest expression levels were found in FAB subtypes M5 and the lowest in M3. In total, increasing levels were shown in the following order: M3 <M3v <M6 <M2 <M4eo <M4 <M0 <M1 <M5a <M5b. Independent analysis of FLT3 expression in different cytogenetic AML subgroups showed the lowest median in the t(15;17) group, followed by t(8;21), inv(16), normal, complex karyotype and the highest median in the t(11q23) group. No difference was observed between the group of secondary AML following MDS, therapy related AML and the de novo AML (p=0.868, p=0.562, and p=0.570, respectively). Compared to clinical parameters, FLT3 expression correlated with high percentage of bone marrow blasts (p=0.0005) and high leukocyte count (p<0.001). In contrast to previous studies no difference in FLT3 expression levels was detected between AML with (n=74) and without (n=130) any FLT3 mutation. Assessment of FLT3 RNA by microarray analysis and FLT3 receptor surface expression (CD135) detected by flow cytometry correlated significantly with FLT3-expression as assessed by real time PCR (p<0.001, each). To analyze whether high FLT3 expression is a prognostic parameter 118 AML cases with normal karyotype were devided into two groups. Group 1 (n=75) was defined to have less and group 2 (n=43) more than the median of the FLT3 expression level found in the total group. No impact on OS and EFS could be shown (608 vs. 311 days, p=0.1283 and 398 vs. 208 day, p=0.3056). In conclusion, these data support the hypothesis that FLT3 activation through mRNA overexpression is an alternative mechanism to FLT3 mutations. Especially as it was found extremely high in 11q23 AML, that rarely reveal FLT3 mutations.