1. The PML Gene Breakpoint in Acute Promyelocytic Leukemia and
Its Association with Clinical and Hematological Characteristics:
A Study of 58 Korean Patients
Mina Hur1,2 , Yoon Hwan Chang1, Ji Yeon Kim1, Hee Jin Kim1,
Dong Young Lee1, Han Ik Cho1, and Sung Sup Park1
1Department of Clinical Pathology, Seoul National University College of Medicine, Seoul, Korea
2Department of Clinical Pathology, Hallym University College of Medicine, Seoul, Korea
INTRODUCTION
PML-RARα rearrangement is the diagnostic hallmark of acute promyelocytic leukemia (APL), and generates heterogeneous PML-RARα fusion transcripts as a result of different breakpoints in the PML gene.
It is still unclear, however, whether the PML-RARα heterogeneity has any biological or clinical relevance.
We analyzed the association of the PML gene breakpoints with clinical and hematological characteristics of APL in Koreans.
MATERIALS & METHODS
Fifty-eight cases of APL were enrolled in this study, who were newly defined at Seoul National University Hospital from January 1995: 53 adults and five children, and 27 males and 31 females.
We determined the PML gene breakpoints using RT-PCR and direct sequencing, and analyzed hemogram, morphologic features, and immunophenotypic expressions according to the PML gene breakpoints (bcr1 and bcr3).
RT-PCR analysis
- Sense primers of the PML gene (PA, PB, and PC) were located in exon 3 (PA and PB) and exon 6 (PC), respectively. Primers RA, RB, and RC were sequences of RARA gene: RA and RB were antisense primers located in exon 3, and RC, sense primer in exon 2.
- Their sequences were :
PA [5'-AGCGCGACTACGAGGAGATG-3'], PB [5'CTGGTGCAGAGGATGAAGTG-3'],
PC [5'-GTCTCCAATACAACGACAGC-3'], RA [5'-CATGTTCTTCTGGATGCTGC-3'],
RB [5'-CCATAGTGGTAGCCTGAGGA-3'], & RC [5'CAGCACCAGCTTCCAGTTAG-3'].
- We used a primer set of PA and RA for the first round PCR, and two sets of primers (PB/RB and PC/RB) for the second round PCR. As RNA internal controls, RA and RC were used for first PCR, and RB and RC, for second PCR.
- Amplification program consisting of denaturation at 94℃, annealing at 55℃, and extension at 72℃ for a total of 35 cycles. Nested PCR was conducted using 1 uL of the primary PCR reaction product in reaction conditions similar to the primary PCR reaction.
- In 14/ 58 cases, sequence analysis was performed to find the exact fusion site.
RESULTS
Breakpoint distribution showed bcr1 in 37 cases (63.8%), bcr2 in two (3.4%), and bcr3 in 19 (32.8%).
The bcr3 type was significantly associated with childhood APL and absence of leukopenia (≥3,500/uL) compared with bcr1 type (P = and 0.03, respectively) (Table I).
Other characteristics including hemoglobin, platelet, morphologic subtype (M3 or M3v), and CD2 or HLA-DR expression showed no significant difference between bcr1 and bcr3 types.
Childhood APL was more frequently associated with M3v type and high white blood cell count compared with adult APL (P = and 0.031, respectively).
DISCUSSION
The association of the PML gene breakpoint patterns with a variety of disease characteristics or prognostic informations has been reported. The focus of previous studies were brought to the issues including age, WBC count, morphologic subtype, and immunophenotypes. We summarized the data of the previous reports and the present study in Table II.
In conclusion, our data shows that bcr3 breakpoint pattern is significantly associated with absence of leukopenia and childhood APL. It is also noteworthy that childhood APL is characteristically correlated with M3v subtype, higher WBC count, and bcr3 breakpoint pattern in these series of Korean patients. This finding supports that age could be associated with the pathogenesis of APL in some ethnic groups.