The potential prognostic value of quantitative real-time reverse transcriptionpolymerase chain reaction (RT-PCR [qrt-PCR]) measurements of PML-RAR␣ mRNA in acute promyelocytic leukemia was retrospectively assessed before treatment and at 3 posttreatment intervals in 123 patients on intergroup protocol 0129. The primary measure was the PML-RAR␣ GAPDH normalized quotient (NQ), that is, PML-RAR␣ mRNA copies divided by glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA copies. Only samples with more than 2.5 ؋ 10 5 copies of the housekeeping gene GAPDH mRNA (detection sensitivity exceeding 10 4 ) were considered NQ evaluable. With RNA from low-density selected cells, paired peripheral blood (PB) and bone marrow samples (n ؍ 140) had comparable NQs (P < .001). Before treatment, high NQ was associated with short-form PML-RAR␣ (P < .001), but not with white blood cell count or clinical outcome. Following treatment, NQ was lower in all-trans retinoic acidinduced complete remission (CR) than chemotherapy-induced CR (P ؍ .018) and at first test after consolidation chemotherapy (P ؍ .037). After consolidation chemotherapy, patients with NQ exceeding 10 ؊5 had 4.1-fold increased relapse risk (P ؍ .008); however, 73% of patients who experienced relapse had NQ lower than 10 ؊5 . In the follow-up period (FUP), any NQ exceeding 10 ؊5 and 10 ؊6 had 17.5-fold and 7.6-fold increased relapse risk, respectively (P < .001), while no gradation of relapse risk (approximately 18%) could be identified at NQ lower than 10 ؊6 , including NQ ؊ .
Of 113 acute promyelocytic leukemia cases documented to have diagnostic PML-RAR alpha hybrid mRNA, 10 cases (8.8%) had fusion sites in PML gene exon 6 (V-forms) rather than in the two common hybrid mRNA configurations resulting from breaksites in either PML gene intron 6 (L- forms) or intron 3 (S-forms). In 4 V-form cases, a common break/fusion site was discovered at PML gene nucleotide (nt) 1685, abutting a 3′ cryptic splice donor sequence. The fusion site was proximal to the common site in 1 case and more distal in 5 cases. The open reading frame encoding a PML-RAR alpha gene was consistently preserved, either by an in-frame fusion site or by the insertion of 3 to 127 unidentified nts. In 2 V-form cases, hybridization analysis of the reverse transcriptase-polymerase chain reaction products with a PML-RAR alpha juction probe was required for discrimination from L-form cases. Two V- form subgroups were defined by in vitro sensitivity to all-trans retinoic acid (tRA)-induced differentiation: 4 of 4 cases tested with fusion sites at or 5′ to nt 1685 (subgroup E6S) had reduced sensitivity (EC50 > or = 10(-7) mol/L), whereas 4 of 4 cases with fusion sites at or 3′ to nt 1709 (subgroup E6L) had high sensitivity (EC50 < 10(-8) mol/L) indistinguishable from that of L-form and S-form cases. These results provide the first link between PML-RAR alpha configuration and tRA sensitivity in vitro and support the importance of subclassifying APL cases according to PML-RAR alpha transcript type.
In addition to the major fusion gene PML-RARα, the t(15; 17) in acute promyelocytic leukemia (APL) produces the reciprocal fusion gene RARα-PML. To determine the scope of RARα-containing mRNA expression in APL cells, we tested PML-RARα–positive APL cells for the presence of mRNAs initiated from two distinct RARα gene promoters, α1 and α2. From the normal allele, both RARα1 and RARα2 mRNAs were expressed in all APL cases (N = 24). From the translocated allele, RARα1-PML mRNA was expressed in 77% and RARα2-PML mRNA in 28% of cases (N = 98). RARα2-PML mRNA was not observed in the absence of RARα1-PML mRNA. There was no association between RARα1-PML or RARα2-PML mRNA expression and the type of PML-RARα mRNA formed by either 5′ or 3′ breaksites in the PML gene. RARα1-PML mRNAs and RARα2-PML mRNAs from 5′ PML breaksite cases coded for full-length RARα-PML proteins but RARα2-PML mRNAs from 3′ PML breaksite cases encoded a truncated RARα2 peptide. RARα1/α2-PML mRNA expression was not associated with differences in APL cell sensitivity to all-trans retinoic acid(tRA)-induced differentiation in vitro or in clinical outcome after tRA or chemotherapy induction therapy (protocol E2491). Our analysis indicated that RARα1/α2-PML mRNA expression markedly differs from normal RARα1/α2 mRNA expression, that the difference in RARα1-PML and RARα2-PML mRNA expression frequency is primarily related to the genomic separation of the RARα1 and RARα2 coding exons, and that variations in RARα1/α2-PML mRNA expression likely have no clinically relevant function in APL cells.
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