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Abstract 1845

Abstract 1270

The p73, a homologue to the tumor suppressor gene p53, is involved in oncogenesis, although its specific role remains unclear. This gene has two distinct promoters which encode for two protein isoforms with opposite transcriptional activities: full-length TAp73 and the ΔNp73, the latter differing by the lack of the N-terminal transactivating domain and, has a dominant-negative function towards p53 and TAp73 activity. In the present study, we have investigated, by Real Time PCR, the gene expression pattern of TAp73and ΔNp73in 147 samples from patients with de novoAML and 22 samples of CD34+ hematopoietic progenitor cells isolated from bone marrow of healthy donors. We detected a significantly higher expression of TAp73and ΔNp73genes in AML samples compared to normal hematopoietic progenitors. AML samples harboring the chromosomal translocations: PML-RARa, AML1-ETOand CBFβ-MYH11presented significantly higher levels of the TAp73than those negative for these rearrangements. In contrast, samples without these gene rearrangements expressed higher levels of the truncated form ΔNp73. No difference was observed among patients with PML-RARa, AML1-ETOand CBFβ-MYH11. In order to determine if the ratio between the relative expression of TAp73 and ΔNp73genes was associated with differential susceptibility to pro-apoptotic stimuli, we performed an in vitrocytotoxicity assay using Ara-C. Blasts from 20 AML patients were incubated for 24 hours in a medium containing 100μg-ml of Ara-C and evaluated for apoptosis status using Anexin V and Propidium Iodite. The leukemic cells with a higher ΔNp73/ TAp73ratio were significantly more apoptosis-resistant (Pearson's R2: −0.607). We also assessed the protein expression by flow cytometry using saturating amounts of specific antibodies for each isoform and analyzing the mean fluorescence channel. Estimated protein expression corroborated the gene expression results. Our results suggest an association between the presence of PML-RARα, AML1-ETOor CBFβ-MYH11chromosomal translocation and a higher TAp73gene expression compared to ΔNp73isoforms expression in AML samples. Moreover, the higher resistence to apoptosis detected in blasts with higher ΔNp73/ TAp73values suggest that this pathway may contribute to prognosis.

The identity and nature of the leukemic stem cell (LSC) is an area of intense research interest since it may lead to a better understanding of the leukemogenic process and development of specific therapies. By definition, the LSC retains the stem cell properties of self-renewal, high proliferative capacity, predominant quiescent cell cycle status and differentiation potential, and is biologically distinct from more differentiated blasts. Here we address the question whether the expression of membrane transporters associated with multidrug resistance (MDR) phenotype is differentially expressed in LSC (defined as CD34+,CD38−CD123+) and more mature blasts CD34+CD38+and the bulk leukemic population. Twenty-one patients with AML de novowere selected based on CD34 positivity by the leukemic blasts. MDR protein expression was measured by flow cytometry with the monoclonal antibodies against P-glycoprotein (P-gp), MDR-related protein (MRP), Lung-resistance protein (LRP) and breast cancer resistance protein (BCRP). In all experiments, at least one hundred thousand events were acquired in a FACScalibur flow cytometer and analysis was performed using the Cell Quest software. The expression levels of MDR transporters were analyzed using:

Acute promyelocytic leukemia (APL) associated with the t(15;17)/PML/RARα and AML with t(8;21)/AML1/ETO are AML subtypes characterized by distinct cytomorphological and clinical features. The coexistence of both genetic abnormalities in a single leukemic clone is extremely rare, and whether it affects PML subcellular distribution or the response to the treatment with all transretinoic acid (ATRA) has not been previously analyzed. Here we report an AML case refractory to treatment in which the PML/RARαor AML1/ETOexpression was analyzed by conventional cytogenetics, FISH, RT-PCR, Southern blot and Spectral Karyotyping (SKY), in addition PML distribution was analyzed by immunofluorescence staining. A 47-year-old female patient complaining of dyspnea for 1 month and presenting hematomas and pallor was referred to the University Hospital of Ribeirao Preto. Adenopathy and splenomegaly were absent. The hemoglobin level was 6g/dl, platelet count was 14x103/ml and leukocyte count was 10.2x103/ml with 22% blasts. The coagulation profile was normal, except for the D-dimers level wich was between 4,000 – 8,000 ng/ml. The differential counts of bone marrow (B.M.) aspirates revealed the presence of 21% blasts and 24% promyelocytes. Immunofluorescence staining of B.M. cytospin preparations using the PG-M3 antibody revealed that PML was delocalizated from the nuclear bodies, a feature suggestive of the diagnosis of APL. The immunophenotypic analysis identified two cell subsets: one CD33+CD13+HLA-DR−CD34−CD15+with high Sideward Scatter (SSC) and another one CD33+CD13+HLA-DR+CD34+CD15−with low SSC values. The karyotype after G-banding was as follows: 46X, iso(X)(q11), t(8;21)(q22;q12) in 18/18 metaphases.SKY analysis confirmed the chromosomal abnormalities detected by G-banding and identified a cryptic insertion of chromosome 15 material into chromosome 17 in 5/5 metaphases.The expression of AML1/ETOand PML/RARαgenes was demonstrated by RT-PCR. FISH analysis were performed using Vysis PML and RARα probes did not detect PML/RARα rearrangements in 300 interphases. On the contrary, FISH assays using Vysis AML1and ETO probes confirmed the presence of t(8;21) in 15% of 300 interphases. Finally, RARα rearrangement was detected by Southern blot analysis performed on B.M. cells genomic DNA using the H18 and K3 RARα genomic probes.The patient was treated with ATRA 45 mg/m2/d for 30 days associated with standard 3+7 AML induction regimen but did not achieve remission. ATRA dose was increased to 90 mg/m2/d and a second identical course of chemotherapy was administered from Day +35. B.M. aspirate obtained on Day +63 presented 2% of blasts/promyelocytes, but the PML/RARαand AML1/ETO transcripts were still detectable by RT-PCR. The patient died of sepsis on Day +67. The lack of response to ATRA observed in this patient contrasts with the favorable outcome observed in the majority of APL patients.Since both PML/RARα and AML1/ETO oncoproteins affect transcription by forming repressor complexes containing histone deacetylase, it is formally possible that their coexpression could lead to irreversible chromatin remodeling. Despite its rarity, the present case is informative because it suggests that PML/RARα and AML1/ETO may synergize and thus render the leukemic cells resistant to treatment.