Double Genetic Mutations in PML-Rara Fusion Gene Confirmed in a Patient Showing Resistance to All-Trans Retinoic Acid and Arsenic-Trioxide Therapy.

Molecular targeting drugs, all-trans retinoic acid (ATRA)and arsenic trioxide (ATO), have major advances in the treatment of acute promyelocytic leukemia (APL). However, resistance to these drugs has been also observed in clinical practice. ATRA acts as a ligand for retinoic acid receptor alpha (RAR) and restores the aberrant transcription repression by PML-RARA fusion protein in APL cells. Previous reports demonstrate that amino-acids substitution, resulting from genetic mutations, in ligand binding domain (LBD) of RARA region of PML-RARA were closely related to drug resistance to ATRA therapy. In contrast, for ATO therapy, the molecular mechanisms of the effectiveness and also the resistance are still unclear. Here we identified a PML-RARA that holds double genetic missense mutations in RARA and PML regions, respectively, from an APL patient, who showed clinically resistance to ATRA and ATO therapy. These mutations were observed as his disease progression, and we are interested in the relationship between these mutations with drug resistance to ATRA and/or ATO.

Analyses of the molecular and clinical significance of the double missense mutations of PML-RARA for disease progression and resistance to ATRA and ATO therapy.

Eight APL patients were treated with ATO in Nagoya University Hospital, Japan, during ∼5 years from Apr. 1, 2000 to Dec. 31, 2004. One out of 8 patients showed clinically ATO resistance. The patient showing ATO resistance firstly diagnosed as APL (M3 variant) from cytogenetic and chromosomal analyses, and complete remission was obtained after combination chemotherapy with ATRA. Molecular CR was confirmed by RT-PCR analysis, but after 3 month from the induction therapy, ATRA-resistant relapse was observed. After treatment with ATO therapy, response was observed, but the effectiveness was gradually decreased, resulting finally into the resistance. The patient died of disease progression. During his 7 years clinical course, leukemia cells were harvested repeatedly from his bone marrow and peripheral blood. RT-PCR using the total RNA from his tumor cells followed by DNA sequencing was performed, with the result of PML-RARA fusion gene with the bcr3 breakpoint in the intron 3 of PML. When using the tumor cells that were harvested at his terminal stage, a missense point mutation in the LBD of the RARA region of PML-RARA was confirmed. Furthermore, missense point mutation in the PML-B2 domain was also confirmed in the same cDNA clones. Interestingly, these mutations were not observed in the leukemia cells obtained at the onset. These mutations were analyzed in each sample that was obtained as his disease progressed, and some correlation between disease progression and/or the drug resistance and the timing of appearance of these two mutations were suggested. These mutated fusion transcripts were cloned into expression vectors, and we are now analyzing the function relating to the drug resistance and disease progression.

Double genetic missense mutations in the RARA-LBD and PML-B2 of PML-RARA were confirmed in ATRA and ATO resistant patient. These genetic mutations were confirmed in the leukemia cells during his disease progression, and the relationship between those mutations and drug resistances were suggested from the clinical features. Mutations in the PML-B2 domain has not been reported previously, thus, it may be important to show whether this type of mutations are related to the drug resistance, especially to ATO therapy.

Kiyoi:Novartis Pharma Co. Ltd.: Research Funding; Kyowa Hakko Kirin Co. Ltd.: Consultancy. Naoe:Kyowa Hakko Kirin Co., Ltd. : Research Funding; Chugai Pharmaceutical Co.,Ltd.: Research Funding; Wyeth K.K.: Research Funding.