Coexpression of PML/RARα and AML1/ETO Leads to ATRA Resistance In Vivo in Acute Promyelocytic Leukemia.

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 trans retinoic acid (ATRA) has not been previously analyzed. Here we report an AML case refractory to treatment in which the PML/RARα or AML1/ETO expression 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/ETO and PML/RARα genes 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.