Identification and monitoring of atypical PML/RARA fusion transcripts in acute promyelocytic leukemia

Abstract

Once the diagnostic suspicion of acute promyelocytic leukemia (APL) has been raised, international guidelines recommend prompt initiation of tailored therapy and supportive care, while awaiting for genetic confirmation of the diagnosis, and the identification of the specific PML/RARA isoform by reverse transcriptase polymerase chain reaction (RT‐PCR). Depending on the PML break point, usually located within intron 6, exon 6, or intron 3, different PML/RARA transcript isoforms may be generated, that is, long (bcr1), variant (bcr2), and short (bcr3), respectively. We report here the characterization of three APL cases harboring atypical PML/RARA transcripts, which were not clearly detectable after standard RT‐PCR amplification. In all three cases, clinical, morphological, and immunophenotypic features were consistent with APL. Direct sequencing allowed the identification of atypical break points within the PML and RARA genes. Then, we designed a patient‐specific quantitative real‐time PCR for the atypical transcripts, which allowed for specific quantitative evaluation of minimal residual disease (MRD) during follow‐up. Despite the rarity of APL cases with an atypical PML/RARA fusion, our study indicates that an integrated laboratory approach, employing several diagnostic techniques is crucial to timely diagnose APL. This approach allows prompt initiation of specific targeted treatment and reliable MRD monitoring in atypical APL cases.

Acute Promyelocytic Leukemia: A Constellation of Molecular Events around a Single PML-RARA Fusion Gene

“…These are characterized by cDNA deletions of either the distal region of PML exon 6 (from 8 to 146 nucleotides), or the entire exon 6, as a result of a mis-splicing event or a genomic break within PML exon 6 (rarely in PML exon 5). Likewise, atypical bcr2 are frequently associated with insertions of three to 127 extra nucleotides (1 to 42 extra amino acids) of genomic DNA from RARA intron 2 [63,[68][69][70][71][72][73][74]. In most of the cases, V-forms are "private", being observed in single APL patients.…”

“…In most of the cases, V-forms are "private", being observed in single APL patients. However, a few cases share the activation of a novel donor splice site in PML exon 6 [72,73], or express a minor PML-RARA transcript with exon 5 skipped [72,74]. Reported isoforms have been compared with the reference sequences of PML and RARA (GenBank accession numbers: NM_033238.2; NM_000964.3).…”

“…Atypical cases of bcr1 with breakpoints located downstream of PML intron 6 have also been reported. In these cases, the donor splice site of exon 7a or 7b is spliced directly with RARA exon 3 [70,77,79], or indirectly through the insertion of genomic DNA (19 to 119 nucleotides) from PML intron 7 or RARA intron 2, respectively [67,74,78,80]. Notably, Yi and colleagues reported a case in which a reverse sequence of 19 nucleotides originated from PML exon 7c complementary sequence was inserted between part of exon 7c (308 nucleotides) and RARA exon 3 [80].…”

“…These rare bcr3 isoforms originate from breakpoints located downstream of PML exon 4, which is then commonly involved in the splicing with RARA exon 3 [73,74,[81][82][83]. In this case, as for bcr2 and bcr1 variants, insertions of genomic DNA sequences might also occur at the junction between PML and RARA genes [74,82,83]. Among others, a short insertion of nine nucleotides (CCCCCAGTT) of unknown origin has been reported in a PML-RARA fusion between PML exon 4 and part of the exon 1 of RARA2.…”

“…The need for more sensitive PCR techniques is driven to improve clinical decision-making. A new alternative method in monitoring MRD is droplet digital PCR (ddPCR), which provides an accurate and highly sensitive absolute quantification of the PML-RARA transcript, including atypical PML-RARA fusion transcripts [74,171]. Using this new method, residual transcripts could be detected independently of the kinetics of molecular relapse, which is APL transcript-specific and not time-dependent [171].…”