atypical mrna fusions in pml-rara positive, rara-pml negative acute promyelocytic leukemia

GENES, CHROMOSOMES & CANCER 49:471–479 (2010)

Atypical mRNA Fusions in PML-RARA Positive,RARA-PML Negative Acute Promyelocytic Leukemia

Christoph Walz,1,2 David Grimwade,3 Susanne Saussele,2 Eva Lengfelder,2 Claudia Haferlach,4

Susanne Schnittger,4 Marina Lafage-Pochitaloff,5 Andreas Hochhaus,2,6 Nicholas C. P. Cross,7

and Andreas Reiter2*

1Pathologisches Institut,Universit�tsmedizin Mannheim,Mannheim,Germany2III.Medizinische Klinik,Universit�tsmedizin Mannheim,Mannheim,Germany3Departmentof Medical and Molecular Genetics,King’s College London School of Medicine,London,UK4Mˇnchner Leuk�mie-Labor,Mˇnchen,Germany5Departmentof Genetics,CHUTimone,AP-HMand Universite¤ de la Me¤ diterrane¤ e,Marseille,France6Department Hematology/Oncology,Universit�tsklinikum Jena,Jena,Germany7Wessex Regional Genetics Laboratory,Salisbury and HumanGenetics Division,Universityof Southampton,Salisbury, UK

Reciprocal RARA-PML transcripts are not detected in �25% of patients with PML-RARA positive acute promyelocytic leuke-

mia (APL), but the reasons for this are poorly understood. We studied 21 PML-RARA positive/RARA-PML negative cases by

bubble PCR and multiplex long template PCR to identify the genomic breakpoints. Additional RT-PCR analysis was per-

formed based on the DNA findings. Three cases were found to have complex rearrangements involving a third locus: the

first had a PML-CDC6-RARA forward DNA fusion and expressed a chimeric PML-CDC6-RARA mRNA in addition to a PML-

RARA. The other two had HERC1-PML and NT_009714.17-PML genomic fusion sequences at their respective reciprocal

breakpoints. Six patients were falsely classified as RARA-PML negative due to deletions on chromosome 15 and/or 17, or al-

ternative splicing leading to atypical RARA-PML fusion transcripts, which were not identified by conventional RT-PCR

assays. This study demonstrates that the frequency of RARA-PML expression has been underestimated and highlights re-

markable complexity at chromosomal breakpoint regions in APL even in cases with an apparently simple balanced

t(15;17)(q24;q12). VVC 2010 Wiley-Liss, Inc.

INTRODUCTION

In acute promyelocytic leukemia (APL; FAB

M3), the vast majority of cases are characterized

by the presence of a t(15;17)(q24;q12) leading to

fusion of the promyelocytic leukemia (PML) genein chromosome band 15q24 and the retinoic acid

receptor alpha (RARA) gene in 17q12 (Grimwade

and Enver, 2004). The reciprocal RARA-PMLfusion gene is not detected in �25% of patients,

including those with an apparently balanced

t(15;17), but the molecular mechanisms why

RARA-PML is absent are largely unknown.

While genomic RARA breakpoints are almost

exclusively found in RARA intron 2, the genomic

breakpoint regions within PML are heterogenous

and clustered within three regions (Figs. 1A and

1B). In 55% of PML-RARA positive patients, the

PML breakpoint occurs within Intron 6 (bcr1) and

results in a PML Exon 6—RARA Exon 3 (Long

(L-) isoform) and a reciprocal RARA Exon 2—

PML Exon 7 mRNA fusion transcript. In 40% of

patients, a breakpoint within PML Intron 3 (bcr3)

gives rise to a PML Exon 3—RARA Exon 3

(Short (S-) isoform) and a reciprocal RARA Exon

2—PML Exon 4 mRNA fusion transcript. In a

minority of patients (5%), a variable (V-) isoform

is created by a PML breakpoint within Exon 6

(bcr2), which is fused with or without the use of

a nucleotide insert to RARA Exon 3. The corre-

sponding reciprocal V-isoform fusion transcript is

formed between RARA Exon 2 and PML Exon 7

due to lack of a specific splice site within the

truncated PML Exon 6.

Because the PML-RARA fusion protein alone

may not be sufficient for full leukemic

Additional Supporting Information may be found in the onlineversion of this article.

Supported by: Deutsche Jose Carreras Leukamie-Stiftung e.V,Germany, Grant numbers: DJCLS R06/02, DJCLS H03/01;German Bundesministerium fur Bildung und Forschung(Projekttrager Gesundheitsforschung; DLR e.V., Grant number:01GI9980/6; European LeukemiaNet funded by the EC LSHC-CT-2004-503216; Leukaemia Research of Great Britain.

*Correspondence to: Prof. Andreas Reiter, III. MedizinischeKlinik, Universitatsmedizin Mannheim, Theodor-Kutzer-Ufer 1-3,68167 Mannheim, Germany. E-mail: [email protected]

Received 14 October 2009; Accepted 14 January 2010

DOI 10.1002/gcc.20757

Published online 12 February 2010 inWiley InterScience (www.interscience.wiley.com).

VVC 2010 Wiley-Liss, Inc.

transformation, it was suggested that one poten-

tial cooperating lesion may be provided by the

reciprocal RARA-PML fusion protein which

increases the penetration (but not latency) of the

leukemic phenotype in PML-RARA transgenic

mice, possibly by deregulating the normal PML

pathway and increasing genomic instability (Pol-

lock et al., 1999; Walter et al., 2007). However,

the exact mechanism by which PML-RARA con-

tributes to disease development is not yet fully

understood since an adapted transgenic mouse

model with lower expression of PML-RARA in

early myeloid cells was found to have a higher

incidence of an APL-like phenotype compared to

earlier transgenic models (Westervelt et al.,

2003). It also remains controversial if there is

indeed a clinically relevant role for the RARA-PML gene as it was shown that the presence of

the reciprocal fusion gene does not affect patient

outcome or complications (Li et al., 1997). How-

ever, the potential presence of cryptic or complex

forward and reciprocal fusion genes has never

been taken into consideration.

We show here that atypical fusion genes can

be detected in many cases that are negative by

conventional PCR assays either as a consequence

of complex rearrangements involving a third gene

or through deletions and alternative splicing

within RARA or PML.

MATERIALS AND METHODS

Patients

Bone marrow (BM) and peripheral blood (PB)

samples from 21 PML-RARA positive/RARA-PMLnegative APL patients at diagnosis were studied.

RNA was available from all 21 patients, genomic

DNA from 20 patients. The patients’ characteris-

tics are summarized in Table 1. Informed consent

was obtained as required by the Declaration of

Helsinki.

Figure 1. Frequently observed genomic breakpoints within PMLand RARA and corresponding fusion transcripts. (A) Commongenomic breakpoint regions within the PML and RARA gene. (B) Thebreakpoint regions bcr1, bcr2, and bcr3 within PML result in two dif-ferent reciprocal mRNA fusion transcripts. While genomic breakwithin bcr3 leads to the S-isoform, a break within bcr1 and bcr2

results in the L-isoform. The truncated PML Exon 6 is not included inbcr2 RARA-PML mRNA due to lack of a splice site; therefore RARAExon 2 is fused to PML Exon 7 as for bcr1 cases. The standardnested RT-PCR primers (R18, RS2, PS1, and SM1) to detect the reci-procal RARA-PML fusion transcript can universally be used for bothisoforms.

472 WALZ ETAL.

Genes, Chromosomes & Cancer DOI 10.1002/gcc

RT-PCR

Details of BM and PB sample preparation,

RNA extraction, cDNA synthesis, and standard

RT-PCR protocols for the detection of PML-RARA and RARA-PML fusion transcripts have

been fully described elsewhere (Grimwade et al.,

1996a,b). For the detection of atypical RARA-PML fusion transcripts, RT-PCR assays were per-

formed including multiple primer pairs derived

from sequences within RARA Exon 1 and

sequences downstream of PML Exon 7. The

sequences of primers are listed in Supporting In-

formation Table 1. All PCR reactions were at

least performed twice. Conventional sequencing

of PCR products was performed with primers

from both, the plus and the minus strand. In

addition, all genomic fusion sequences obtained

by the bubble PCR approach were verified

by specific, individually designed primer sets

and subsequent sequencing of the amplification

product.

Multiplex Long-Template PCR and Bubble-PCR

For the detection of forward PML-RARA and re-

ciprocal RARA-PML genomic junction sequences

in individual patients, a combination of multiplex

long-template PCR (MLT-PCR) and bubble PCR

was performed essentially as described (Zhang

et al., 1995; Reiter et al., 2003). Intronic forward

and reciprocal primers were selected from sequen-

ces flanking the putative genomic breakpoints

within PML and RARA as indicated by the junc-

tion sequence at the cDNA level. For MLT-PCR,

several forward primers upstream of RARA Exon 2

and covering the entire region of RARA Exon 1

and Intron 1 (R1I, R1II) were used in combination

with diverse reverse primers derived from

genomic sequences downstream of PML Exon 4

(P5I, P5II) and Exon 7 (P8I, P8II), respectively, in

single reactions (Fig. 1). Amplified products were

sequenced either directly or after cloning using

the TOPO cloning kit (Invitrogen, Leiden, The

Netherlands).

TABLE 1. Patients’ Characteristics from 21 Patients with PML-RARA Positive/RARA-PML Negative APL

No. Age KaryotypeTranscript

typePML-RARAmRNA

PML-RARAgDNA

RegularRT-PCR forRARA-PML

RT-PCR forRARA-PMLwith newprimer sets

RARA-PMLgDNA

1 13 n.a. S PML-RARA PML-RARA neg. r1p4 r1p42 33 n.a. L PML-RARA PML-RARA neg. r2�p7 r2�p73 75 t(15;17),del (9q) S PML-RARA n.a. neg. r1p8 n.a.4 18 t(15;17) S PML-RARA PML-RARA neg. r1p7 r1p75 65 t(15;17) S PML-RARA n.a. neg. Dr2p4 n.a.6 75 t(15,17) complex S PML-RARA PML-RARA neg. r1-ins-p4 r2p47 34 t(15;17) S PML-RARA and

PML-CDC6-RARAPML-RARA andPML-CDC6-RARA

neg. neg. neg.

8 39 t(15;17);þ8 L PML-RARA PML-RARA neg. neg. neg.9 52 t(15;17),del9(9q) S PML-RARA PML-RARA neg. neg. neg.

10 49 t(15;17) L PML-RARA PML-RARA neg. neg. neg.11 62 n.a. S PML-RARA PML-RARA neg. r1p4 r1p412 25 46,XX,del(15)(q?)[8]/46,XX[11] L PML-RARA HERC1-PML neg. neg. neg.13 22 46,XY,add(12)(p1?),-13,

add(15)(q22),add(17)(q12),þmar

L PML-RARA NT_009714.17-PML neg. neg. neg.

14 n.a. n.a. L PML-RARA PML-RARA neg. neg. neg.15 56 46,XY,t(15;17)(q22;q11) S PML-RARA PML-RARA neg. neg. neg.16 32 46,XX L PML-RARA PML-RARA neg. neg. neg.17 49 46,XX,-8,þt(8;?)(p23;?),

t(15;17)(q22;q12)L PML-RARA PML-RARA neg. neg. neg.

18 50 46,XY,t(1;17;15)(p36;q21;q22)[7]/46,XY[2]

L PML-RARA PML-RARA neg. neg. neg.

19 43 46,XX,del(9)(q?),t(15;17)(q22;q11)[12]/46,XX[7]

L PML-RARA PML-RARA neg. neg. neg.

20 43 46,XX,t(15;17)(q22;q11)[10] S PML-RARA PML-RARA neg. neg. neg.21 20 46,XX,t(15;17)(q22;q11)

[19]/46,XX[1]S PML-RARA PML-RARA neg. neg. neg.

MOLECULAR MECHANISMS UNDERLYING RARA-PML NEGATIVE APL 473


RESULTS

We identified 21 patients that tested positive

for PML-RARA but negative for the reciprocal

RARA-PML fusion transcript by conventional RT-

PCR assays. The median age was 43 years (range,

13–75). Further clinical details on treatment and

response are available for 20 of 21 (95%) patients.

All patients received ATRA in addition to con-

ventional chemotherapy. Two patients died

within few weeks due to ATRA-syndrome. All

other patients achieved complete hematologic

remission and are alive after a median observa-

tion time of 119 months (range, 1–154). To

explore the reasons for RARA-PML negativity, we

first determined the forward genomic PML-RARAjunction sequences in 19 cases by MLT-PCR

and bubble PCR; in one of the remaining two

cases no genomic DNA was available and in the

other case PCR amplification was unsuccessful.

In 17 patients, PML was found to be directly

fused to RARA at the genomic level, while in

three cases sequences of a third gene were iden-

tified within the junction region (Figs. 2A–2C).

Identification of Complex Rearrangements

Involving a Third Gene

Patient no. 7 displayed a PML-CDC6-RARAfusion gene at the genomic level with expression

of an in-frame PML-CDC6 (truncated Exon 6)-

RARA mRNA fusion in addition to a ‘‘normal’’

PML-RARA fusion transcript (Fig. 2A, Supporting

Information Fig. 1C). CDC6 is located immedi-

ately upstream of RARA on 17q12 suggesting a

submicroscopic deletion of 50-RARA-sequences.No reciprocal RARA-CDC6, CDC6-PML, or RARA-PML fusion sequences could be amplified at the

mRNA or DNA level suggesting an even more

complex rearrangement. Patient no. 12 showed a

complex reciprocal genomic fusion gene involving

a sequence derived from Intron 33 of the HERC1gene (which is located upstream of PML in

15q24) fused to PML Intron 6 exactly at the

genomic breakpoint which was identified by char-

acterization of the forward PML-RARA junction

sequence (Fig. 2B). Of interest, cytogenetic anal-

ysis showed a 46,XX,del(15)(q?)[8]/46,XX[11]

while RT-PCR amplified a normal PML-RARAfusion transcript (L-isoform). The deletion could

therefore be a del(15)(q22q24), while the appa-

rently normal chromosome 17 may harbor a sub-

microscopic insertion of 17q12 sequences

including 30-RARA into 15q22. Unfortunately

adequate material was not available to confirm

this by fluorescence in situ hybridization.

In patient no. 13, bubble PCR with an antisense

primer derived from a genomic PML-sequence that

was located immediately downstream of the forward

genomic PML breakpoint amplified a sequence in

which PML was fused to a genomic sequence

derived from chromosome 12p (NCBI accession

number NT_009714.17) (Fig. 2C). This genomic

fusion was confirmed with a sequence-specific

primer combination. Cytogenetic analysis revealed

a 46,XY,add(12)(p1?),-13,add(15)(q22),add(17)(q12),

þmar. Despite the intervening 12p sequence iden-

tified at the genomic level, a normal PML-RARAfusion transcript was detected by RT-PCR.

Atypical RARA-PML Fusion Genes due to

Deletions on Both Derivative Chromosomes

In three patients, large deletions of 10, 12, and

23 kb, respectively, involving RARA Exon 2 and

parts of Intron 1 upstream of the forward

genomic RARA Intron 2 breakpoint were identi-

fied (Fig. 3). As a consequence, atypical RARA-PML fusion transcripts were generated in all

three cases at the mRNA level fusing RARAExon 1 to PML Exon 4 (r1p4, n ¼ 2) or PMLExon 7 (r1p7, n ¼ 1) as shown by RT-PCR (Fig.

3A). Additional atypical RARA-PML fusion tran-

scripts were identified in two patients by RT-

PCR with newly designed primer sets (R1I-II

and P8I-II, Fig. 1B) taking into account the

potential occurrence of deletions leading to loss

of conventional primer binding sites (r1p8; Dr2p4,deletion of 299 bp; Fig. 3B).

Alternative Splicing of RARA Exon 2

RT-PCR with forward primers derived from

RARA Exon 1 and reverse primers from RARAExon 4 revealed a fusion transcript with an inter-

vening stretch of 147 bp which was derived from

RARA Intron 2 (r1-ins-p4, n ¼ 1) (Fig. 3C). The

genomic RARA breakpoint was located immedi-

ately downstream of the inserted sequence within

RARA Intron 2. The inserted sequence was pre-

ceded by a cryptic ‘‘AG’’ splice site and followed by

a ‘‘GT’’.

Overall, among the 21 cases analyzed, 5 differ-

ent atypical RARA-PML fusion transcripts were

identified in six patients: r1p4 (n ¼ 2); Dr2p4(n ¼ 1); r1p7 (n ¼ 1); r1p8 (n ¼ 1); and r1-ins-p4

(n ¼ 1) in PML-RARA positive patients who were

previously suspected RARA-PML negative by

conventional RT-PCR (Fig. 4). In the remaining

15 cases, use of the primer sets derived from

474 WALZ ETAL.


RARA Exon 1 and PML Exons 5 and 8, respec-

tively, did not yield any fusion transcripts. The

fusion sequences of the atypical PML-RARA/

RARA-PML mRNA transcripts are shown in Sup-

porting Information Figures 1 and 2, respectively.

Sequence chromatograms of the atypical RARA-

Figure 2. Creation of complex fusion genes with involvement of additional genes: (A) PML-CDC6-RARA in patient no. 1, (B) HERC1-PML in patient no. 2 and (C) PML fused to a sequence derived fromchromosome 12p.



PML mRNA transcripts are shown in Supporting

Information Figure 3.

DISCUSSION

In PML-RARA positive APL, we show here that

apart from insertion events (Grimwade et al., 2000),

RARA-PML negativity as determined by conven-

tional RT-PCR assays can be the consequence of at

least three independent mechanisms: (i) complex

genomic fusion sequences including a third gene,

(ii) deletions on one or both derivative chromo-

somes, and (iii) alternative splicing ofRARAExon 2.

Figure 3. (A) Large deletions (up to 23 kb) of sequences upstream of the forward genomic breakpointof RARA led to loss of primer binding sites which are usually located within RARA Exon 2 and to the for-mation of atypical RARA-PML fusion transcripts. (B) Generation of an atypical RARA Exon 1—PML Exon 8fusion transcript. (C) Alternative splicing leading to a RARA Exon 1—ins—PML Exon 4 fusion transcript.

476 WALZ ETAL.


Our study reveals that the generally accepted

estimates of 20–25% RARA-PML negativity in

PML-RARA positive APL is largely overesti-

mated. Small deletions on both derivative chro-

mosomes seem to be quite common. If these

deletions are large enough, they can result in loss

of primer binding sites which are usually selected

from sequences derived from RARA Exon 2 lead-

ing to false negative results. In our series of 21

patients, some deletions led to expression of

atypical RARA-PML fusion transcripts in a signifi-

cant proportion (6/21, 29%) of patients. Interest-

ingly in such cases, PML sequences of variable

length are fused to RARA Exon 1 which entirely

contains the 50-UTR of the RARA gene. Conse-

quently, the fusion gene only contains coding

sequences of the truncated PML gene, and it is

unclear whether any protein product would be

produced. Similarly, some of the other atypical

fusions we observed are out of frame and thus

may not to be translated. However, it is worth

noting that the regions encoding all known

domains of PML (the RING and B-Box zinc fin-

ger domains, nuclear localization signal, Pro-rich

domain, and potential coiled-coil domain) are lost

in the r1p7 and r1p8 transcripts. With the excep-

tion of the nuclear localization signal domain, all

these domains are also lost in the r1p4 transcript.

Several cases have been reported with the

involvement of a third translocation partner in

addition to the breakpoints in Chromosomes 15

and 17 (Borrow et al., 1994; Brunel et al., 1996;

Wan et al., 1999) and indeed many instances of

complex variants of other recurrent translocations

in AML have been described. However, only a

few of these have been characterized at the

genomic level. Several reciprocal translocations

are known to be associated with submicroscopic

deletions of sequences on the derivative chromo-

somes, e.g., t(12;21)—ETV6-RUNX1, t(8;21)—

RUNX1-RUNX1T1 (AML1-ETO) or those invol-

ving MLL at 11q23 (Kolomietz et al., 2001;

Meyer et al., 2006; Moon et al., 2007), but most

extensively studied in t(9;22)(q34;q11) in chronic

myeloid leukemia (CML) involving BCR and

ABL1 genes. However, the results regarding the

prognostic value of these deletions have been

inconsistent (Huntly et al., 2001; Kolomietz et al.,

2001). Recently, Kreil et al., reported no differ-

ence in overall survival when patients with or

without deletions on the derivative Chromosome

9 were compared (Kreil et al., 2007). However,

the subgroup of patients with deletions spanning

the ABL1-BCR breakpoint had a significant in-

ferior prognosis while patients with deletions only

on either the BCR or ABL1 side of the breakpoint

had a superior prognosis. The biological basis of

these effects remains to be determined.

With the exception of the MLL-recombinome

(Meyer et al., 2006), studies with comparable case

numbers are not available for other translocations.

Structural alterations including duplications, dele-

tions, and amplification of ETV6 or RUNX1 were

reported as favorable prognostic factors in ETV6-RUNX1 positive childhood acute lymphoblastic

leukemias (Martinez-Ramirez et al., 2001). Stan-

dard G-banding (resolution: �5–10 Mb), FISH

(resolution: �100 kb), and array-based compara-

tive genomic hybridization (resolution: �5–10 kb)

(aCGH) identified submicroscopic deletions on

the der(15) or der(17) in only a very small number

of APL patients with t(15;17) (5/198; 3%) (Laf-

age-Pochitaloff et al., 1995; Kolomietz et al., 2001;

Specchia et al., 2002; Bacher et al., 2005; Subra-

maniyam et al., 2006; Moon et al., 2007; Dolan

et al., 2008). Although some authors have linked

these deletions with an adverse prognosis, case

numbers were low and clinical follow-up data

were not available for all patients. The presence

of deletions of RARA and/or PML identified by

FISH analysis in two APL patients was recently

linked to potential resistance to ATRA-based

therapy (Subramaniyam et al., 2006). In the first

patient in this study, a deletion of �800 kb

encompassed a major part of the 30 end of PMLand the entire 50 end of RARA on der(17)t(15;17)

including Exons 1 and 2. In the second patient,

an estimated �150-kb deletion encompassed the

amino terminus of PML located 50 of Exon 6 with

retention of signals upstream of the PML gene.

Our data revealed no significant differences in ini-

tial response rate to ATRA-based chemotherapy

Figure 4. Atypical RARA-PML fusion transcripts at mRNA level inpatients previously reported RARA-PML negative by standard RT-PCR.



and relapse-free or overall long-term survival as

compared to RARA-PML positive patients sug-

gesting that the characterized structural alterations

as submicroscopic deletions are possibly without

prognostic significance in PML-RARA positive

APL.

In three cases the reciprocal fusion gene prod-

uct was not created because of complex rear-

rangements at the genomic breakpoint junction

region with involvement of genes from distant

genomic regions. One patient with a classical

t(15;17) by conventional cytogenetics revealed a

novel chimeric fusion gene involving CDC6,which is located immediately upstream of RARAon 17q12. RT-PCR revealed coexpression of an

in-frame PML-CDC6 (Exon 6)-RARA mRNA

fusion in addition to typical PML-RARA fusion

transcripts. CDC6 is implicated in regulation of

DNA replication, with Exon 6 encoding the AAA

ATPase domain raising the possibility that the

novel triple fusion gene could have contributed

to the disease in this patient (Takahashi et al.,

2002). Characterization of a case with del(15)(q?)

revealed a fusion of HERC1, a gene which is

located upstream of PML on 15q24, to PMLIntron 6. HERC1 is a giant multidomain protein

located in the cytosol and in the golgi apparatus

that interacts with ARF1 and RAB proteins and

may act as guanine nucleotide exchange factor

and E3 ubiquitin ligase (Garcia-Gonzalo et al.,

2003). No samples were available to look for the

expression of these atypical fusions at the protein

level. A third case with complex karyotype

revealed a fusion of the genomic 30-sequences of

PML to a sequence derived from 12p. No

genomic sequences fused to the 50-sequences of

RARA could be determined indicating a very

complex translocation. Advanced technologies as

whole genome sequencing or paired-end

sequence mapping might be helpful to clarify the

nature of these complex rearrangements in the

future (Collier and Largaespada, 2006; Dempsey

et al., 2006; Chen et al., 2009).

In 12 of 21 cases (57%), no atypical RARA-PML transcripts could be characterized and there

was no evidence for complex rearrangements

because PML-RARA fusion genes at the DNA

and mRNA levels were normal in all cases. Based

on the identification of deletions on both deriva-

tive chromosomes in a significant proportion of

cases, we speculate that genomic breakpoints

may be located even more proximal to RARA or

distal to PML due to even larger deletions which

may only be detectable by FISH or aCGH.

The potential pathogenetic role of the recipro-

cal fusion protein for APL disease pathogenesis

was also demonstrated in PLZF (ZBTB16)-RARApositive APL which is associated with primary re-

sistance to ATRA treatment. Transgenic PLZF-RARA positive mice developed a leukemic di-

sease lacking typical features of APL but more

reminiscent of CML-like disease (He et al.,

1998). In contrast, a model expressing PLZF-RARA in combination with its reciprocal counter-

part RARA-PLZF resulted in a leukemia with dis-

tinct features of APL such as the block of

differentiation at the promyelocytic stage (He

et al., 2000). This difference in phenotype was

most likely caused by interference of the RARA-

PLZF oncoprotein with the normal PLZF path-

way since transgenic PLZF-RARA/PLZF�/�

mice also developed a leukemia similar to APL

and indistinguishable compared to the disease

observed in the PLZF-RARA/RARA-PLZF

model. Normal PLZF was shown to act as a tran-

scriptional repressor of the gene encoding the ret-

inoic acid binding protein CRABPI (Guidez

et al., 2007). In RARA-PLZF positive APL blast

cells, the normal function of PLZF is abrogated

leading to an upregulation of CRABPI and

increased retinoid resistance.

In conclusion, this study reveals considerable

heterogeneity of genomic breakpoint regions at

the derivative Chromosomes 15 and 17 in PML-RARA positive APL. Because atypical RARA-PMLfusion genes are found in a considerable propor-

tion of patients, we recommend that PCR proto-

cols for the detection of RARA-PML should be

modified by inclusion of primers corresponding to

RARA Exon 1 rather than RARA Exon 2. Further

studies are warranted to investigate the extent to

which pathogenesis and phenotype of APL is

influenced by these heterogeneities.

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atypical mrna fusions in pml-rara positive, rara-pml negative acute promyelocytic leukemia

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