Leukemia (1998) 12, 1866–1880
 1998 Stockton Press All rights reserved 0887-6924/98 $12.00
http://www.stockton-press.co.uk/leu
REVIEW AND MEETING REPORT
Acute promyelocytic leukemia: a curable disease
F Lo Coco1, C Nervi2, G Avvisati1 and F Mandelli1
1
Department of Cellular Biotechnology and Hematology, and 2Department of Histology and Medical Embryology, University ‘La Sapienza’,
Rome, Italy
The Second International Symposium on Acute Promyelocytic
Leukemia (APL) was held in Rome in 12–14 November 1997.
Clinical and basic investigators had the opportunity to discuss
in this meeting the important advances in the biology and treatment of this disease achieved in the last 4 years, since the First
Roman Symposium was held in 1993. The first part of the meeting was dedicated to relevant aspects of laboratory research,
and included the following topics: molecular mechanisms of
leukemogenesis and of response/resistance to retinoids, biologic and therapeutic effects of new agents such as arsenicals
and novel synthetic retinoids; characterization of APL heterogeneity at the morphological, cytogenetic and immunophenotypic level. The updated results of large cooperative clinical
trials using variable combinations of all-trans retinoic acid
(ATRA) and chemotherapy were presented by the respective
group chairmen, and formed the ‘core’ part of the meeting.
These studies, which in most cases integrated the molecular
assessment of response to treatment, provided a stimulating
framework for an intense debate on the most appropriate frontline treatment options to be adopted in the future. The last day
was dedicated to special entities such as APL in the elderly
and in the child, as well as the role of bone marrow transplantation. The prognostic value of molecular monitoring studies
was also discussed in the final session of the meeting. In this
article, we review the major advances and controversial issues
in APL biology and treatment discussed in this symposium and
emerging from very recent publications. We would like to credit
the successful outcome of this meeting to the active and generous input of all invited speakers and to participants from all
over the world who provided constructive and fruitful
discussions.
Keywords: acute promyelocytic leukemia; PML/RAR␣; all-trans
retinoic acid
Molecular mechanisms of retinoid and arsenic actions in
APL
APL is uniquely associated with chromosomal translocations
that disrupt the gene encoding for the retinoic acid receptor ␣
(RAR␣) at 17q21.1–5 In more than 99% of cases, this disruption
results in the fusion of RAR␣ with the PML gene of chromosome 15q22. Rare variants of APL have been described, in
which three novel RAR␣ fusion partners were identified: the
PLZF (promyelocytic leukemia zinc finger) gene on chromosome 11,6 the nucleophosmin (NPM) gene on chromosome
57 and the NuMA (nuclear mitotic apparatus protein) gene on
Correspondence: F Lo Coco, Dept of Cellular Biotechnology and
Hematology, University ‘La Sapienza’, Via Benevento 6, 00161 Rome,
Italy; Fax: 39 6 44 24 19 84
This Review is also a Meeting Report as it is based on an extension
and updating of the literature relating to the data presented at the
Second International Symposium on APL (Acute Promyelocytic Leukemia: A Curable Disease), Rome, 12–14 November 1997 (Chairman:
Professor Franco Mandelli).
Received 21 August 1998; accepted 4 September 1998
chromosome 118 (Table 1). PML, PLZF, NPM and NuMA are
not structurally related, but are nuclear proteins that contain
an N-terminal protein–protein interaction domain. RAR␣ is
one of the nuclear retinoid receptors that mediate action of
ATRA on myeloid differentiation.9–12 When fused to RAR␣,
the resulting chimeric proteins possess an identical RAR␣ moiety (B-F regions of the receptor), comprising the DNA, corepressor, coactivator and ATRA binding regions. The occurrence of a unique RAR fragment fused to multiple partners
suggests that RA-target genes are downstream effectors of their
activities.1–5
APLs with the PML/RAR␣, PLZF/RAR␣, NPM/RAR␣ or
NuMA/RAR␣ are clinically and phenotypically very similar
but differ in their sensitivity to ATRA. In particular, APL
patients and cells expressing PLZF/RAR␣ fail to differentiate
in response to ATRA.13
Expression of both PML/RAR␣ or PLZF/RAR␣ into hematopoietic precursor lines induces a differentiation block and promotes survival and both PML/RAR␣ and PLZF/RAR␣ transgenic mice develop an APL-like disease.14–20 This leukemia is
ATRA-sensitive in PML/RAR␣ transgenic mice and relatively
ATRA-resistant in PLZF/RAR␣ mice. Accordingly, PML/RAR␣,
but not PLZF/RAR␣, increases ATRA sensitivity of hematopoietic precursor cells.
Thus, both fusion proteins appear actively involved in conferring a differentiation block to leukemic cells, while only
PML/RAR␣ confers ATRA sensitivity.14,15 ATRA probably converts PML/RAR␣, but not PLZF/RAR␣, to an active inducer of
myeloid differentiation possibly by triggering its transcriptional activator function on specific ATRA target genes.
Notably, structural and functional alterations of PML/RAR␣
may cause ATRA resistance in APL-derived NB4 cell
lines.21–24
Retinoic acid receptors
Retinoids, natural or synthetic derivatives of vitamin A, are
necessary dietary constituents that exert dramatic effects on
development, cell proliferation and differentiation by regulating the expression of specific genes.9,10,24 Biological
effects of retinoids appear to be mediated by two classes of
nuclear receptor proteins of the steroid and thyroid hormone
superfamily, the retinoic acid receptors (RARs) and the retinoid X receptors (RXRs). RARs (␣, ␤ and ␥) are activated by
both ATRA and 9-cis-RA, whereas RXRs (␣, ␤ and ␥) are activated by 9-cis-RA only.9,10 As the other members of this superfamily, retinoid receptors are ligand-inducible transcriptional
regulatory factors and RARs must heterodimerize with RXRs
for effective DNA binding and transactivation.9,10 It has
recently been shown that RARs associate with co-regulatory
proteins, such as N-CoR and SMRT, in the absence of ligand.
These repressors, in turn, associate with Sin3 and histone
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F Lo Coco et al
Table 1
Chromosome breakpoint
Involved gene
Putative function
15q22
PML
Transcription factor
11q23
PLZF
Transcription factor
5q32
NPM
Nucleolar phosphoprotein
11q13
NuMA
a
1867
RAR␣ partner genes and fusion proteins associated with an APL phenotype
Nuclear mitotic apparatus protein
Hybrid product (isoforms)
Response to ATRA
PML/RAR␣
(bcr1-bcr2-bcr3)
PLZF/RAR␣
(A and B)
NPM/RAR␣
(L and S)
NuMA/RAR␣
yes
no
?a
yes
The single published case of NPM/RAR␣-positive APL was recently shown to be ATRA-reponsive in vitro at relapse.81
deacetylase proteins to form a transcriptional repressor complex.24,25 Ligand binding is proposed to dissociate the
repressor complex and to recruit a multisubunit activation
complex that possesses histone acetyltransferase activity.25
The PML gene product
PML is a member of the RING finger family that is ubiquitously expressed in tissues within large multiprotein nuclear
structures termed nuclear bodies (NBs) and acts as a growth
suppressor in vitro.26–29 PML interacts and co-localizes with
PIC-1, an ubiquitin-like molecule also called SUMO-1 (for
small ubiquitin-related modifier) that was isolated as a yeast
two hybrid partner of PML.30 The characterization of ubiquitin-like (UBL) genes and their biological roles was the subject
of Solomon’s presentation. E Solomon (London, UK) proposed
that PIC-1 or SUMO-1 should be called in the future UBL-1,
for ubiquitin like-1 protein. UBL-1 is highly homologous to a
S. cerevisiae gene called SMT3, which is believed to be
important for normal DNA replication, nuclear division and
mitotic spindle integrity.31 The isolation of mouse UBL-1
cDNA revealed an homology of nearly 100% of the predicted
amino acid sequence between mouse and human. This
extreme conservation stresses the importance of this gene family. The mouse UBL-1 gene was found to map to chromosome
1, which should correspond to chromosome 2 in humans. A
number of mouse and human pseudogenes were pulled out
during the isolation of the genomic sequence.32
Immunofluorescence studies show that UBL-1 co-localizes
with PML into the PML-NBs and that the immunostaining patterns of both UBL-1 and PML are disrupted and appear diffuse
in APL cells. After ATRA treatment, the PML and UBL-1 proteins entirely co-localized.30 In addition, human and murine
UBL-1 were found covalently linked to other proteins such as
human DNA repair proteins (RAD51 and RAD52), the cell
death domain of FAS and a Ran GTPase-activating protein
(Ran-GAP1) that is required for functional nuclear transport.31,32 These data suggest that conjugation with UBL-1
might regulate PML degradation or transport into the nucleus
or its interaction with the nuclear matrix.
Nuclear localization of PML
A Dejean (Paris, France) presented work aimed to link the
therapeutic effect of ATRA in promoting myeloid cell differentiation to its effect in reorganizing the nucleus. Immunofluorescence studies have shown that the native PML protein concentrates in the nucleus within discrete nuclear structures
identified as a subtype of nuclear bodies.26–28 The PML bodies
correspond to a new type of multiprotein complex in which
two components (PML and SP100) have been genetically
characterized to date. The expression of PML and SP100, as
well as the number of NBs, are strongly up-regulated by treatment with interferon ␣.33 These structures can be disrupted by
environmental stimuli, such as heat shock and viral proteins
(herpes simplex, adenovirus, CMV). Thus, these structures
could correspond to active sites for some nuclear functions or
to storage compartments for cellular regulatory proteins. Since
the expression of PML/RAR␣ in APL cells alters the PML-NBs
and their restoration correlates with disease remission, their
function is a key question. PML/RAR␣ could exert a dominant
negative effect by sequestering and diverting a number of proteins from their natural site of action (including RXR and other
nuclear co-factors). Thus, the therapeutic effect of ATRA in
promoting myeloid cell differentiation could be related to the
drastic reorganization of the nucleus which occurs in APL
cells after ATRA treatment.
Molecular mechanisms of APL leukemogenesis and
response to ATRA
ATRA treatment induces a degradation of PML/RAR␣ fusion
protein34,35 an event that has been proposed to account for
the induction of APL cells differentiation by ATRA. Downregulation of PML/RAR␣ by ATRA occurs at the post-translational level,35 and is prevented by proteasome inhibitors.34
A possible role for the proteasome pathway in the ATRA
induced degradation of PML/RARa is indicated by the fact that
PML is associated with ubiquitin-related proteins.30–32,36 However, the proteasome has been found to be involved in cellular
pathways leading to apoptosis due to caspase activation.37,38
Caspases are a family of cysteine proteases with aspartic acid
substrate specificity, thought to be key effectors of cellular
apoptosis in multicellular organisms.39
C Nervi (Rome, Italy) showed that caspases and, in particular, a caspase3-like activity, mediate the ATRA-induced
degradation of PML/RAR␣.40 Using different caspase inhibitor
peptides, the ATRA-induced PML/RAR␣ degradation could be
prevented and the contribution of PML/RAR␣ degradation and
PML-NBs reorganization to ATRA response of APL cells
directly tested. The results show that in PML/RAR␣-expressing
cells, including blasts from APL patients, the extent of
PML/RAR␣ cleavage directly correlates with the ability of
ATRA to restore the normal PML nuclear bodies (NBs) pattern.
However, ATRA-induced APL cell differentiation is not prevented (but is actually increased), by the persistence of the
fusion product and can occur also in the absence of normally
structured PML-NBs.40
H de Thé (Paris, France) presented data that the
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F Lo Coco et al
1868
microspeckled pattern, seen in PLZF/RAR␣-expressing primary
blasts and transfected cells (using either ␣-PLZF or ␣-RAR
antibodies) disappears after overnight exposure to ATRA.
Thus, ATRA fails to induce terminal differentiation of
PLZF/RAR␣-APL blasts despite apparent degradation of the
PLZF/RAR␣ fusion protein.15 Together these findings show that
the degradation of the chimeric proteins is not a crucial event
for the differentiative response of APL cells to ATRA and that
ATRA-induced differentiation does not require the integrity of
the PML-NBs.
PG Pelicci (Milan, Italy), A Dejean (Paris, France) and WH
Miller (Montreal, Canada) presented data that both PML/RAR␣
and PLZF/RAR␣ are associated with a transcription corepressor complex that is involved in both the development
of APL and in the ability of patients to respond to retinoids.20,41–45 Their studies indicate that, in the absence of ligand,
both PML/RAR␣ and PLZF/RAR␣ (similarly to wild-type RAR␣)
are tightly bound to the transcriptional co-repressors N-CoR
and SMRT through the hinge region of the RAR␣ moiety.
SMRT and N-CoR associate with the co-repressor Sin3 and
histone deacetylase proteins to form a transcriptional repressor
complex.46,47 Thus, binding of PML/RAR␣ and PLZF/RAR␣ to
transcriptional co-repressors leads to the formation of a
repressive chromatin structure. The affinity of the interaction
between these two oncoproteins and transcriptional corepressors is much higher than that of wild-type RAR␣ but
appears critical for their leukemogenetic activity. In fact, PG
Pelicci showed that the mutation of the region of PML/RAR␣
which binds N-CoR causes the loss of the ability of PML/RAR␣
to block differentiation, without affecting its capacity to
increase ATRA-induced differentiation in vitro.43 Indeed,
much higher ATRA concentrations are required for transcriptional corepressors to dissociate from PML/RAR␣ than from
wild-type RAR. In contrast, two distinct N-CoR/SMRT interaction regions are present in the PLZF/RAR␣ fusion. One in the
COR box of the RAR␣ moiety (that is shared with RAR␣ and
PML/RAR␣) and another in the PLZF moiety. ATRA addition
fully releases the complex interacting with the RAR␣ component of PLZF/RAR␣, but not that interacting with the PLZF
moiety, which appears totally resistant to ATRA. Notably,
inhibition of the repression block mediated by the histone
deacetylase enzymatic activity by treatment with Trichostatin
A (TSA) or sodium butyrate is able to synergize with ATRA
to convert PLZF/RAR␣ from a repressor of transcription to an
activator of ATRA target genes and myeloid differentiation.
WH Miller presented data showing that TSA also induces a
partial restoration of the ATRA-induced differentiation in RAresistant NB4-R4 cell lines, in which a point mutation in the
PML/RAR␣ E domain causes a loss of ligand binding (see
below).23,44
Together, these studies20,41–45 demonstrate that the inability
of APL-fusion proteins to dissociate from the nuclear corepressor complex at physiological ATRA concentrations is
critical for PML/RAR␣ and PLZF/RAR␣ leukemogenic activity.
The different stability of the fusion/corepressor complex in the
presence of pharmacologic levels of ATRA is responsible for
their differences in ATRA sensitivity.
Retinoid-resistance
To study the molecular basis of the acquired resistance to
ATRA that occurs in APL patients following treatments with
ATRA alone, WH Miller developed stable-retinoid resistant
subclones of the APL cell line NB4.22,23 These cell lines retain
the t(15;17) and appear to be resistant also to a variety of retinoids including those that do not seem to bind CRABP. Immunohistochemical studies indicate that in retinoid-resistant NB4
subclones, ATRA treatment fails to alter the microparticulate
nuclear pattern of PML.21–23 These subclones present altered
retinoid binding properties probably due to an abnormal
PML/RAR␣ expression, structure and function.21–23 In fact, the
DNA binding activity and the stimulation of transcription of
an RARE driven CAT-reporter gene induced by ATRA is significantly decreased in these cells compared to the NB4 parental cell line.22,23 In addition, a partial loss of retinoidinduced gene expression in these cells is present. In one of
these subclones (R4) a lysine:proline point mutation in the
ligand binding domain of the RAR␣ portion of the fusion protein was found. This proline substitution causes loss of ligand
binding of PML/RAR␣, but no loss of binding to RAREs or
nuclear co-repressors.23,44 A similar mutation was detected in
APL blasts from three ATRA-resistant patients by Gallagher et
al.48 More recently, Imaizumi and co-workers described the
acquisition of point mutations in the ligand binding region of
RAR␣/E-domain in two patients who exhibited ATRA-resistance at relapse.49 Such mutations, which resulted in amino
acid changes (Arg:Gln and Met:Leu, respectively) in the affected domain, were associated with decreased ligand-dependent transcriptional activity of the mutant PML/RAR␣ protein
as compared to that of wild-type PML/RAR␣. Thus, the resistant phenotype and mechanism found in cell lines in vitro may
be paralleled by a very similar mechanism in APL patients.
Molecular basis of clinical response to arsenic trioxide
Arsenic trioxide has been identified as a potent antileukemic
agent in both ATRA-sensitive and ATRA-resistant APL
patients.50,51 It acts by triggering apoptosis without differentiation of APL blasts.50–55 Similarly to ATRA, in APL cells
arsenic trioxide rapidly induces a drastic reorganization of the
nucleus characterized by the restoration of intact PML NBs
that appear increased in size, followed by a progressive degradation of the PML/RAR␣ protein.50,54,55 Thus, both ATRA and
arsenic may induce re-assembly of PML-NBs as a consequence of the induction of PML/RAR␣ degradation.52,55 WH
Miller showed that arsenic trioxide induces apoptosis, reassembly of PML-NBs and loss of PML/RAR␣ also in a large
panel of ATRA-resistant NB4 cell lines.55 Indeed, studies on
fresh APL cells in vitro show that the addition of arsenic interferes with the capacity of ATRA to induce cellular differentiation, while the addition of ATRA reduces cell apoptosis
induced by arsenic.55 In contrast, data presented by H de Thé
indicate a cooperative effect on differentiation and apoptosis
by the combined treatment with arsenic and ATRA in arsenic
resistant NB4 subclones.52 Notably, fresh blasts from a
t(11;17) APL patient were found to be totally unresponsive to
arsenic treatment in vitro. Further studies of the mechanism
of action of these two agents may better define their respective
roles in the treatment of APL and, possibly, of other tumors.
The restoration of PML-NBs by arsenic is not confined to APL.
In non-APL cells, arsenic does not induce significant degradation of PML, but rather triggers its post-translational attachment to SUMO-1 (also called PIC-1, and proposed by E Salomon to be renamed UBL-1).36 This subsequently re-targets
PML from the nuclear matrix to the nuclear bodies. In fact,
experimental evidence presented by A Dejean and H de Thé
indicate that arsenic induces a very fast movement of the 90
kDa unmodified PML from the nuclear soluble fraction to the
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F Lo Coco et al
nuclear matrix, where SUMO-1 oligo- and poly-modified
forms of PML are associated with components of the PML
NBs. The formation of these modified SUMO-1-PML conjugated forms (which is a phosphorylation-dependent process),
increases considerably after arsenic treatment.36 Together
these data suggest that post-translational modifications by
SUMO1 are involved in the trafficking of proteins between
different subcellular structures and provide additional evidence that ubiquitin-like modifications are not restricted to
degradation signals.
Modern approach for integrated diagnosis of APL:
advantages and pitfalls of the various techniques
Given its unique response to a specific therapy, correct identification of APL is of the utmost importance in the clinical practice.1–5,56,57 Moreover, early institution of ATRA therapy seems
effective in the control of the APL-associated coagulopathy,
which accounts for the frequently recorded early hemorrhagic
deaths and whose prompt correction is therefore essential for
successful outcome.58–63 A diagnostic approach should therefore be outlined which defines the impact of each test in either
therapeutic choice or prognostic assessment.
Most recent characterization data summarized in the following paragraphs indicate that each technique carries its
own pitfalls and advantages. To properly tackle the issue of
diagnostic assessment, it is appropriate to distinguish two
groups of procedures: (1) those mandatory for patient entering
into tailored (ie ATRA-containing) treatment; and (2) those
useful for better dissecting potentially distinct categories
and/or for monitoring minimal residual disease. For therapeutic decision, the crucial point is cytogenetic or molecular
demonstration of the unique t(15;17) abnormality, which is by
no doubt the condicio sine qua non for eligibility into ATRAcontaining protocols. As first demonstrated in 1992 by Miller
et al,64 RT-PCR allows better definition of this aberration and
clearly defines patients responsive to ATRA. In fact, among
cases with apparently normal metaphases or non-evaluable
karyotypes, only those with RT-PCR detectable PML/RAR␣
responded to ATRA.64 This observation was further confirmed
in several larger studies.65
It is now universally accepted that PML/RAR␣ is the real
hallmark of this disease, as it may be found in the absence of
t(15;17).1–5,65–71 Therefore, it is important to emphasize that
negative t(15;17) does not preclude a diagnosis of APL. For
example, in the MRC ATRA study, only 87% of patients with
molecular evidence of APL had the t(15;17) (failures and cryptic translocations accounting for the remaining 13%).72 Demonstration of the t(15;17) by conventional karyotype or FISH,
however, is specific for APL and PML/RAR␣ expression.
Identification of microspeckled PML distribution by anti-PML
monoclonal antibodies used in immunocytochemistry or
immunofluorescence provides an equally specific approach to
allow patient entry into tailored protocols if RT-PCR evaluation is not available.73,74 Compared to RT-PCR, the analysis
of PML localization offers the additional advantage of rapidity
(2–3 h), low cost, and simplicity of execution/interpretation.73,74 Thus, its use might be recommended in small
centers not equipped or trained for molecular studies. However, it is worth emphasizing that RT-PCR is the only approach
allowing precise definition of the PML/RAR␣ isoform (long or
bcrl-2 vs short or bcr3). This, in turn, provides a target for
minimal residual disease evaluation, and the definition of the
appropriate RT-PCR strategy (including the correct primer set)
to be used for the individual patient during follow-up.65
The MRC, GIMEMA and PETHEMA groups found that
approximately 10% of cases morphologically defined as M3
in multicenter trials lacked the specific fusion gene after RTPCR.72,75,76 It is important that, in cases with typical M3 morphology, treatment is not delayed until genetic characterization is available. In fact, unless performed on-site, the
execution of cytogenetic and RT-PCR analyses may require
several days, particularly in large cooperative studies in which
diagnostic material is centrally reviewed. On the other hand,
cases with clear evidence of PML/RAR␣ by RT-PCR should
always be further characterized with complete karyotyping on
banded metaphases for diagnostic confirmation and for the
identification of potentially relevant additional abnormalities.
Finally, immunophenotype has an important role, as we will
discuss below, due to its capability of identifying in AML a
surface marker profile consistently associated with (although
not unique for) APL. In cases with uncertain morphology (M3v
and others) certain immunophenotypic patterns are extremely
useful for fostering further characterization, in particular genetic demonstration of the specific abnormality. Whether distinct APL immunophenotypic subsets bear prognostic significance is the subject of presently ongoing investigation.
Advantages and pitfalls of diagnostic characterization techniques used in APL are shown in Table 2.
Diagnostic and differentiation-induced cytological features
Because recognition of the specific genetic lesion appears
mandatory for patient eligibility into ATRA-containing protocols, in the past few years the role of molecular biology for
diagnostic assessment has become increasingly relevant. Notwithstanding, morphologic characterization of this entity
remains fundamental. Firstly, more than 90% of true APLs (ie
genetically confirmed) are easily and rapidly diagnosed upon
light microscopy based on the characteristic cytological features. Secondly, morphologic analysis is relevant to the identification of less typical variants (microgranular type M3v, basophil-like M3) which may mimic other FAB subsets.
However, cases of suspect M3v77–80 showing scarcely
granulated blasts, strong myeloperoxidase positivity, bilobed
or folded nuclei and monocytoid aspect should be immediately referred to molecular biology and/or cytogenetic laboratories for search of the specific genetic abnormality. In the
clinical practice, one of the key diagnostic issues is when to
alert molecular biologists for RT-PCR of the PML/RAR␣ hybrid
in AML. In other words, the question is: given a possible false
negative result of karyotyping (eg poor mitoses or occurrence
of a cryptic translocation) and the time required, should we
search for the presence of PML/RAR␣ in all AML cases regardless of FAB definition? In our experience (University La Sapienza, Rome), none of 100 non-M3 AML analyzed retrospectively by Southern blot showed rearrangement of the RAR␣
gene. On the contrary, as we have mentioned before, approximately 10% of patients initially classified as FAB M3 and
registered in the GIMEMA trial on the basis of morphology
alone were subsequently excluded due to lack of PML/RAR␣
in leukemic cells.75 In this context, it is important to consider
that translocations other than t(15;17) involving the RAR␣
locus (ie t11;17, t5;17) may result in an APL-like phenotype.6–8
Although extremely rare, t(11;17)-APL needs to be distinguished from PML/RAR␣-positive cases due to distinct
therapeutic requirements.13 As to the t(5;17), which involves
the nucleophosmin (NPM) gene on 5q35 and RAR␣ on
17q21,7 a recent report indicated that this subset is responsive
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F Lo Coco et al
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Table 2
Diagnostic characterization of APL
Methods
Morphology
Immunophenotype
Karyotyping
PML-staining
Markers
Time required
Dysplastic promyelocytes or M3v blast
appearance
CD13+ CD33+ CD9+ CD15s+ CD117+
CD34± TdT− HLA-DR−
t(15;17)
30 min
Southern blot
PML-protein distribution pattern
(microspeckled vs nuclear bodies)
RAR␣ and PML rearrangement
RT-PCR
PML/RAR␣ fusion gene
to ATRA in vitro.81 More detailed information on either forms,
as well as on the recently described t(11;17) involving the
NuMA on 11q13,8 would be needed for a better biologic and
clinical characterization of these rare entities.
Another area of morphological research deserving special
attention is the study of differentiation pathways during retinoic acid therapy. Baseline and ATRA-induced morphologic
aspects of APL were discussed in the symposium by G Flandrin (Paris, France) and V Liso (Bari, Italy). Morphological signs
of response usually start appearing after the first week of
ATRA, and consist of a decrease of cytoplasmic granules and
basophilia, condensation of nuclear chromatin, appearance of
cytoplasmic vacuolization. These features are accompanied
by progressive band-like aspect of the nuclei in the following
days.62 Recognition of these morphological changes is a fundamental criterion for patient initial response to ATRA, though
they may show some variability, also depending on the concomitance and type of combined chemotherapy. Interestingly,
patients receiving identical ATRA-containing treatments may
show heterogeneous morphological changes. For example, in
patients treated with simultaneous idarubicin and ATRA, the
post-induction phase is accompanied in most patients by marrow aplasia, but a non-hypoplastic pattern is also observed in
some others. The reasons for this heterogeneous morphologic
behavior appear unclear and deserve tailored studies correlating these aspects with molecular features and prognosis. Also,
it remains to be established whether a morphologic pattern of
rapid differentiation (ie within 20–30 days) is associated with
better outcome with respect to slow maturation pattern.
Karyotypic variants and cryptic translocations
R Berger (Paris, France) reviewed karyotypic aspects of APL
and proposed some technical recommendations for improving
the rate of successful karyotyping. These include marrow in
vitro culture (up to 48 h), which is less likely than direct preparations to result in the analysis of normal elements (eg
erythroblasts) not belonging to the leukemic clone.82 In recent
years, cytogenetic studies have contributed the identification
of numerous variant translocations including relatively frequent rearrangements of 15, 17 and a third chromosome (ie
chomosomes 1, 8, 14 and others,83–87 and more rare translocations involving RAR␣ on chromosome 17 and PLZF chromosome band 11q23 or NPM from chromosome 5q35.6,7
Most recently, the fusion between the NuMA gene on
2–3 h
24–48 h
2h
4–8 days
6–10 h
Specificity
Major drawbacks
++
M3v and the ATRA-resistant t(11;17)
(poorer in M3v) forms hardly recognized
++
Same phenotype is sporadically found
in ATRA-resistant non-M3 AML
++++
Poor quality of mitoses, cryptic
translocation (frequent false negatives)
+++++
Lack of information on the PML
rearrangement type
++++
Laborious and time-consuming. RAR␣
rearrangement alone may not result
from t(15;17)
+++++
Requires highly qualified expertise
Frequent poor RNA yield
chromosome band 11q13 and RAR␣ has been described as a
single case report.8
Important additional information, which may only be provided by standard karyotyping, is the existence of secondary
changes (trisomy 8, and others) apart from t(15;17). These are
found in approximately 15% to 30% of cases.1–5 Although
their prognostic significance is still uncertain, it seems that
such additional lesions are not associated with unfavorable
outcome as they are, for example, in other AML subsets and in
CML.88 Fluorescence in situ hybridization (FISH) with specific
RAR␣ and PML probes used either in metaphase or interphase
preparations may add important information as to the existence of masked translocations in cases with apparently
normal karyotype.69,71
A Biondi (Monza, Italy) reported the preliminary results of
a European workshop on M3-like leukemias without the
classical t(15;17), which were reviewed morphologically and
analyzed by a combination of RT-PCR, Southern blot and
FISH. At the cytogenetic analysis, this series included cases
with normal karyotype, cases with the t(11;17) and cases
involving chromosome 15, 17 and a third chromosome.
Results of molecular analyses with RT-PCR and FISH showed
the frequent occurrence of cryptic PML/RAR␣ translocations
formed as a result of insertion, whereas all the t(11;17) cases
had the PLZF/RAR␣ fusion gene as expected. The occurrence
of cryptic PML/RAR␣ fusion as the most frequent finding in
morphologically typical APL lacking the t(15;17) was reported
in the symposium and in a recent study by D Grimwade
(London, UK).71 Importantly, these cases showed the characteristic microparticulate pattern of PML distribution when analyzed with a specific antibody, indicative of disruption of the
nuclear bodies, whereas all t(11;17) cases displayed the
typical non-APL pattern of PML localization into discrete
nuclear bodies.71
These studies clearly contradict earlier cytogenetic reports
claiming that a t(15;17) could be found in every APL patient,89
and emphasize the need of combining molecular and karyotypic characterization for diagnostic purposes. The final analysis of large number of cases enrolled into cooperative trials
(US Intergroup, European APL ’93, GIMEMA-AIDA, MRCATRA, PETHEMA LAP’96, JALSG-AML92 and others), which
in most cases involve multiparametric diagnostic characterization, should provide relevant information about the incidence and clinical significance of variant and cryptic translocations in APL.
Review
F Lo Coco et al
Immunophenotypic features
E Paietta (Bronx, New York) discussed the utility of immunophenotyping in APL. Known distinctive features of this entity
include the lack or low expression of HLA-DR and of CD34,
combined to positivity for CD13, CD33 and CD9.90–94 More
recently, absence of the multidrug resistance mediator P-glycoprotein (P-gp) and expression of CD117 and CD15s have
been reported.95 Interestingly, such surface marker pattern
remains consistent in the two main subtypes M3 and M3
microgranular variant (M3v), in spite of their morphological
heterogeneity. Although highly distinctive, this antigenic profile is neither unique nor diagnostic, and cases showing this
phenotype but lacking the specific genetic lesion have been
reported.91 Nonetheless, surface marker analysis is relevant for
diagnostic identification of cases with uncertain morphology
(ie M3v or others) which deserve molecular search of
PML/RAR␣. Other antigens infrequently expressed on APL
blasts are CD2, CD34 and CD19.94 Among these, CD2 positivity has been variably associated with the M3v subset or with
the short (bcr3) PML/RAR␣ transcript type.96,97 By studying
molecular and immunophenotypic features of both children
and adults with APL, we recently found that CD2 is associated
with FAB M3v in the pediatric population and with bcr3 in
adults.94 Thus, it is conceivable that the higher incidence of
CD2 expression described in children reflects the higher
frequency of M3v.
A recently described immunophenotypic subset, the NK (for
natural killer)-cell myeloid leukemia characterized by CD56
expression, may be misdiagnosed as APL upon morphologic
analysis.98 As remarked by E Paietta, this and other immunophenotypic subsets showing only subtle differences compared
to classical APL deserve special attention and require molecular investigation for the presence of the specific genetic lesion.
Despite these limitations, which clearly indicate that lack of
absolute specificity of the surface marker profile, immunophenotypic characterization is of considerable help for diagnostic studies, particularly in light of its rapidity, widespread
availability and low cost.
A further advantage of surface marker analysis is the potential to differentiate discrete clinical entities such as, for
example, CD34-positive or CD2-positive APL. Most recently,
a small subset of patients who co-expressed the B cell marker
CD19 has been reported in association with high white blood
cell count, ie an established negative prognostic factor in
patients with APL.94
The APL-associated coagulopathy in the ATRA era
Early hemorrhagic deaths due to the associated coagulopathy
were recorded in up to 30–40% of APL patients prior to the
use of ATRA.1–5,99 Presenting clinical features and laboratory
parameters indicating a hemorrhagic diathesis with rapid consumption of platelets and coagulation factors were typically
worsened with the start of chemotherapy, resulting in
life-threatening complications including intracranial bleeding.1–5,99 Because the disease has otherwise a favorable prognosis, overcoming the hemorrhagic risk has long since been
considered crucial for successful outcome.
The introduction of ATRA in the front-line therapy has
improved the control of APL-associated coagulopathy. This
impact was discussed in the symposium by T Barbui
(Bergamo, Italy) and extensively reviewed in the
literature.58–63
The pathogenesis of this coagulopathy is rather complex
and can involve several mechanisms including activation of
the coagulation, fibrinolysis, and non-specific proteolysis systems, as demonstrated by modern laboratory assessment. Activation of the coagulation is revealed by elevated thrombinantithrombin (TAT) complexes, prothrombin fragment 1 + 2
and fibrinopeptide A (FPA) plasma levels, whereas high
fibrinogen-fibrin degradation products (FDP), D-dimer and
urokinase-type plasminogen activator (u-PA) levels with concomitantly decreased plasminogen and ␣-2-antiplasmin indicate hyperfibrinolysis. Finally, elevated leukocyte elastase and
fibrinogen split products of elastase testify to the activity of
non-specific proteases. Thrombocytopenia due to either bone
marrow invasion or platelet consumption following intravascular coagulation is found in the vast majority of patients at
presentation.59,63
The improvement of clinical and laboratory signs
accompanying this coagulopathy was soon observed in earlier
studies employing ATRA alone for remission induction.58,59
Usually, decrease of fibrinolysis products and clotting activation markers is detected within the first week of therapy.
Such improvement of laboratory parameters is accompanied
by prompt resolution of associated clinical symptoms.
Notably, such benefits are also observed in patients receiving
simultaneous chemotherapy in addition to ATRA.60–63 The differentiation process which follows ATRA administration
reduces significantly the expression of procoagulant activity
from APL blasts and may partially explain the amelioration of
symptoms related to the coagulopathy. In addition, ATRA acts
through the synthesis of u-PA inhibitors by APL blasts, thus
downregulating cellular plasminogen activator activity. Also,
it protects the endothelium from the prothrombolytic stimulus
exerted by the inflammatory cytokines (TNF-␣, IL-1␤) produced by APL blasts. ATRA also interacts with the hemostatic
system by increasing expression of TM (the surface high-affinity receptor for thrombin) on endothelial cells, resulting in
stimulation of anticoagulant activity. Further, ATRA inhibits
procoagulant activity of monocytes and regulates adhesion
molecules expression on leukemic cells influencing their
adhesion to the vascular endothelium.63 In summary, ATRA is
thought to exert an anticoagulant action that results from a
combination of effects on leukemic, endothelial and monocytic cells.
At the clinical level, the impact of this agent in reducing
early hemorrhagic deaths is still uncertain, as up to 10% of
fatal hemorrhagic events were recorded in most recently
reported trials.72,75,100–107 In particular, the results of two randomized studies comparing ATRA plus chemotherapy vs
chemotherapy alone (European APL ‘91)100 and ATRA vs
chemotherapy (US Intergroup)106 showed a tendency to a
reduction of hemorrhagic deaths in patients given ATRA that
did not reach statistical significance.
Front-line therapy
The results of recently conducted large clinical trials in newly
diagnosed APL were presented by R Warrell (MSKCC, New
York, USA), P Fenaux (for the European APL Group), G Avvisati (for the GIMEMA Group, Italy), M Tallman (for the US
Intergroup), R Ohno (for the JALSG, Japan), A Burnett (for the
MRC, UK) and MA Sanz (for the PETHEMA Group, Spain).
The design of each study and the results obtained are summarized in Table 3. Data from another recently published experience (MD Anderson Group), which were not reported in the
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Table 3
Group
Upfront treatment of APL: results of recent trials
Study design
European (APL’93) ATRA→/+CHTa
MSKCC (New York) ATRA→CHT
MDACC (Houston)
ATRA+IDA
MRC (UK)
ATRA→/+CHTb
GIMEMA (Italy)
ATRA+IDA
Intergroup (USA)
CHT vs ATRAd
JALSG (Japan)
ATRA→CHT
PETHEMA (Spain)
ATRA+IDA
Randomized Pt No. Confirmed
genetic
diagnosis
yes
no
no
yes
no
yes
no
no
403
30
43
239
480
346
196
100
93%
100%
95%
90%
100%
64%
NAe
100%
CR
Early Resistance ATRA
deaths
syndrome
89–95%
7%
0.2%
87%
17%
0
77%
19%
4%
70–87% 23–12% 9–2%
93%
7%
0.3%
69–72% 14–11% 17–17%
88%
9%
0
87%
11%
2%
15%
23%
26%a
3–1%c
9%
26
6%
16%
Outcome
Ref.
2 yrs EFS, 63–80%
Median RFS ⬎28 m
1.5 yrs DFS, 80%
4 yrs DFS, 59–74%
4 yrs DFS, 75%
3 yrs DFS, 32–67%
4 yrs DFS, 62%
too early
107
101
104
72
108
106
105
76
The APL ’93 study compared ATRA given sequentially (→) or concurrently (+) to CHT; CR and EFS in this trial include results obtained in
patients ⬎66 years (lower values) up to the best arm (ATRA+CHT) in patients ⬍66 years.
a
ATRA syndrome in this trial is referred to as ‘suspected’.
b
The MRC trial compared a short 5 day course of ATRA given prior to CHT vs a more extended course commenced simultaneously with CHT.
c
This % includes here only deaths from ATRA syndrome.
d
The Intergroup study compared ATRA vs CHT.
e
APL diagnosis was genetically confirmed in the majority of patients in the JALSG trial.
symposium, have been included in the Table. Overall, more
than 1800 newly diagnosed APL patients were enrolled in
these trials.72,76,101,104–108
Induction
Following the preliminary results with ATRA obtained in
China109 and successively reproduced in Europe110 and the
USA,111 the advantage of using this agent in the front-line therapy was clearly established in early 1993 by the APL European Group who reported the results of a randomised study
(APL-91)100 comparing ATRA followed by chemotherapy
(ATRA→CHT) with chemotherapy alone (CHT). In light of the
significantly better relapse-free survival in the former group,
the CHT arm was terminated early.100 The results of successive studies, which in most cases aimed at determining the
optimal ATRA plus CHT combination (sequential or
ATRA→CHT, simultaneous or ATRA+CHT), clearly indicate
better results in patients who are given ATRA+CHT. Patients
included in these trials (Table 3) had CR rates of 72–95% and
3–4 years DFS rates of 62–75% with better figures obtained
in those receiving the simultaneous combination (Refs
72,101,104–108 and Sanz MA, personal communication).
Noteworthy, resistant leukemia was minimal or absent in studies where all patients had diagnosis confirmed at the genetic
level (by karyotype and/or RT-PCR). Three trials76,104,108 in
which idarubicin was used as a single CHT agent together
with ATRA provided further support to the notion that Ara-C
may be omitted from induction therapy in this disease.112–115
Additional advantages of the ATRA+CHT combination include
a better control of both ATRA syndrome and coagulopathy,
the two most severe complications previously observed at
high frequency using ATRA and CHT as separate agents.1–5
In addition, prompt administration of steroids at the earliest
symptoms of the syndrome, as recommended by the New
York MSKCC Group,101,116 proved highly effective to prevent
the development of an ‘overt’ syndrome. No more than 1–3%
of deaths attributable to ATRA syndrome were recorded in
these trials.72,76,101,104–108
Consolidation
At least two cycles of intensive post-remission therapy were
employed in all studies and currently represent the recommended approach for consolidation. These courses should
mandatorily include anthracyclines, whereas the benefit of
Ara-C is still unclear. Preliminary data from the MD Anderson104 and PETHEMA76 groups suggest that Ara-C may be
omitted not only from induction but also from consolidation,
though long-term results of these trials are required to clarify
this issue. No studies have as yet attempted to compare different consolidation options in phase III trials and the role of AraC certainly represents one of the most important points to be
addressed in the future. The vast majority of patients (up to
95%) are reported to achieve molecular remission – ie convert
to PCR-negative for the PML/RAR␣ fusion gene – at the end
of consolidation in the Italian,108 British,72 Spanish76 and New
York MSKCC117 series.
Maintenance
It has been proposed that APL, unlike other AML, may benefit
from some type of maintenance therapy. In particular, two
studies conducted prior to the ATRA era suggested the advantage of employing methotrexate (MTX) and 6-mercaptopurine
(6-MP) as continuous or intermittent therapy after consolidation.113,118 In light of these data, and taking into account a
potential role of ATRA in prolonging the DFS, the European
APL ’93 and the Italian GIMEMA trials randomized patients
into four maintenance arms after the end of consolidation, ie
MTX and 6-MP, ATRA alone (given intermittently, for 15 days
every 3 months), ATRA + CHT, or observation.107,108 Preliminary results of both trials indicate an advantage of employing
some type of maintenance. A better outcome in terms of DFS
for patients receiving maintenance ATRA was also reported
by the US Intergroup, although the continuous administration
of retinoid therapy in this study was associated with significantly greater toxicity.106
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F Lo Coco et al
Prognostic factors
Specific subsets: APL in children and in the elderly
Despite the significant improvement in survival achieved with
the advent of ATRA + CHT combinations, a sizable proportion
of APL patients still die of their illness.1–5,56,57 Efforts aimed at
ameliorating disease outcome include three main investigational areas, ie (1) improved control of the hemorrhagic syndrome; (2) identification of adverse prognostic features at presentation in order to better adapt the intensity of post-remission
therapy in distinct risk-groups; and (3) early identification and
rescue of recurrent disease (discussed below).
The impact of several clinical and biological parameters on
CR rate and survival was analyzed in most of the clinical studies presented at the symposium. All investigators agreed that
younger age and lower WBC at presentation were prognostic
factors for longer EFS. As regards cut-off values, most studies
considered 30 years for age and 10 × 109/l for WBC. The UK
MRC group showed there to be little difference between using
5 or 10 × 109/l as the appropriate cut-off for WBC.72 In the
JALSG trial, absence of purpura at diagnosis, together with
younger age and lower WBC was an additional favorable factor for CR achievement.105 By contrast, several other biological features such as the type of PML/RAR␣ isoform, additional
karyotypic abnormalities, and expression of the reciprocal
RAR␣/PML transcript had no influence on either CR rate or
overall survival.72,75,101,104–107 A less favorable outcome for
patients with the short PML/RAR␣ isoform, already described
in a study of the MSKCC group,119 was observed in the
GIMEMA trial, but the difference with patients with the long
transcript form was not statistically significant (unpublished
observations). Similarly, a recent study of the US Intergroup
reported the absence of prognostic significance of the two
major PML/RAR␣ fusion types.120 In a previous report, however, the same group observed a decreased response to ATRA
in vitro in patients showing the rarest bcr2 (also referred to as
‘variable’) PML breakpoint type.121 Importantly, the UK MRC
study72 demonstrated that a slower kinetics of PML/RAR␣
negativization was, together with presenting WBC greater than
10 × 109/l, an independent predictor of adverse prognosis. In
fact, detection of residual disease by RT-PCR immediately following the third consolidation course predicted an increased
relapse risk associated with poorer overall survival (AK Burnett
and D Grimwade, UK).72 The prognostic impact of biological
and clinical factors in APL patients receiving ATRA and CHT
is summarized in Table 4.
APL has been variably reported as being more or less frequent
in children than in adults, mainly depending on different geographic areas. For instance, the frequency of the disease seems
much higher in children living in Italy than in those living in
the USA.122,123 In addition, a higher prevalence of the
microgranular type have been described in the pediatric
population.80,122 Indeed, epidemiological studies that take
into account potential bias due to misdiagnosis (eg lack of
genetic validation, which is crucial in the M3v subtype) are
needed to confirm these observations. Phenotypic and molecular features of APL in adult vs pediatric patients have been
compared in a recent study presented in the symposium.94
This analysis was conducted by multiparameter diagnostic
characterization in a series of 196 cases with a molecularly
confirmed PML/RAR␣-positive APL, including 133 adults and
63 children aged ⬍15 years. In children, the disease was
characterized by statistically significant more frequent incidence of the M3v subtype and of short PML/RAR␣ isoform or
bcr3, and by higher WBC. CD2 was directly correlated with
the M3v form in children and with bcr3 in adults.94
A further difference observed in children is related to ATRA
toxicity. Due to reported side-effects in the CNS, and particularly the occurrence of pseudotumor cerebri at higher frequency in children,124 lower ATRA doses (ie 25 mg/m2) have
been given to patients aged ⬍20 years in the ongoing Italian
GIMEMA trial.75 According to a preliminary study conducted
in France,125 such lower doses are equally effective, although
it is not yet clear whether they are associated with less ATRArelated toxicity.
Besides adjusting ATRA dosage, the therapeutic approach
for pediatric APL patients should be the same as that
employed for adult cases. Retrospective studies122,123 analyzed by G Rivera (Memphis, USA) and data on children
extrapolated from the US Intergroup106 and the Italian
GIMEMA108 studies indicate that pediatric patients have similar response to treatment and overall outcome with respect to
non-elderly adults when treated with CHT or modern ATRAcontaining regimens.
Prior to the advent of ATRA, elderly patients with APL had
a dismal prognosis, much like the one of age-matched patients
with other AML subtypes.56 In addition to poorer tolerance
to intensive therapy, these patients may show more frequent
adverse biological features such as hyperleukocytosis and
multidrug resistance P-170 protein and/or CD34 expression.
However, no studies have as yet analyzed in detail the characteristics of genetically proven APL in the elderly, nor have
these features been compared to those of younger patients.
The introduction of ATRA in the front-line therapy and its
combination with CHT has considerably improved the
prognosis of elderly patients with APL. Only one study has
been reported to date which specifically analyzes clinical
outcome in patients aged more than 60.126 Together with data
on the elderly population pooled from most recent
trials,72,75,101,104–108 this study clearly shows a dramatic
improvement in terms of either early mortality and relapsefree survival rates in patients receiving variable combinations
of ATRA and CHT. Therapeutic results on 60 patients aged
⬎60 enrolled in the Italian trial ‘AIDA’ were presented by MC
Petti (Rome, Italy), who underlined the important progress
achieved with this protocol. The results (85% CR and 57% 3year EFS rates) compared favorably with respect to the preATRA era. As expected, preliminary analysis of clinical out-
Table 4
Prognostic factors in APL patients receiving ATRA and
chemotherapy
Favorable variables
Age ⬍30 years
No purpura
WBC ⬍10 × 109/l
Rapid PML/RAR␣
clearance during
consolidation
a
Not significant/
Uncertain
PML breakpoint
Additional cytogenetic
abnormalities
RAR␣/PML
expression
RT-PCR status after
induction
Unknown
Truncated PML
Additional molecular
abnormalities
Unfrequent
immunophenotypes
Others?
Data derived from Refs 72, 75, 88, 94, 100–108, 119, 120.
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F Lo Coco et al
1874
come in this age subset also showed a reduced tolerance to
consolidation therapy.
Therapy of relapse, new drugs and role of hemopoietic
stem cell transplantation
The synthetic retinoic Am80 has been investigated by the
Japanese group who showed a 58% CR rate in patients
relapsed after ATRA.127 In light of the long first CR duration
in this cohort (median time: 22 months), it might be argued
that ATRA would have been effective as well in these patients.
However, this compound deserves further investigation and
molecular and clinical data on a longer follow-up are currently awaited. Another retinoic derivative, 9-cis RA, has been
employed at the MSKCC in New York for relapsed APL.128
Unlike ATRA, 9-cis RA is able to activate both the RARs and
RXRs pathways.9,10 However, preliminary data showed that,
despite showing a more favorable pharmacokinetic profile, 9cis RA was unable to overcome resistance in cases previously
treated with ATRA.128 It remains to be established whether
there is any advantage in combining 9-cis RA, instead of
ATRA, to anthracycline-containing CHT.
A number of pre-clinical studies have shown that certain
arsenic (As) derivatives are effective against APL blasts in
vitro.50,52–55 At the clinical level, one study has been reported
which suggests that As03 is a highly promising therapeutic
strategy for APL in light of its specificity and limited toxicity.51
Here again, no data are as yet available concerning response
at the molecular level and remission duration in these
patients. The putative mechanisms of action of As03, which
mainly acts as an apoptotic inducer,50–55 suggest a potential
important role of this agent given as consolidation after cytotoxic drugs and/or differentiating agents, as discussed in the
symposium by Z Chen (Shanghai, China). Preliminary data on
refractory APL treated with As03 should be made available
soon by the Shanghai and New York groups.
Given the outlook of newly diagnosed patients treated with
combined ATRA+CHT, there is currently no indication for
either allogeneic or autologous bone marrow transplantation
(BMT) in first CR. Data from the EBMT registry, reviewed in
the symposium by A Bacigalupo (Genova, Italy), currently
provide only scarce information on recently treated patients
and are mainly related to the pre-ATRA era.129 For patients
receiving autologous BMT in second remission, a leukemiafree survival of 31% has been reported in the last published
analysis, but this report did not take into account the PCR
status of grafted marrow.129
Approximately 20 to 30% of patients treated with up-front
state of art approaches have been reported to relapse.1–5,56,57,72,75,101–107 Although their outlook is reasonably
optimistic, relapsed APL patients are at higher risk of early
death and more frequently show refractory disease or a shortlived second remission.1–5,56,57 First CR duration and, particularly, ability to undergo a second PCR-negative remission
after reinduction are crucial determinants for successful outcome. As a general rule, these patients were considered candidates for aggressive therapy followed, when applicable, by
autologous or allogeneic bone marrow transplantation.
Second CR may be achieved with ATRA ± CHT in most cases,
provided that first CR duration is ⭓8 months.5,56,57 Presence
of detectable leukemia by RT-PCR after reinduction and consolidation should foster the choice of most aggressive
approaches such as allogeneic BMT or experimental therapies
using newly developed and potentially non-cross-resistant
agents. For patients who achieve second molecular remission,
autologous BMT (or peripheral blood stem cell infusion) is an
effective and widely applicable approach.130
Because of the promising, but yet scarce information
presently available on the newly developed approaches
(AM80, 9-cis RA, As03, etc), treatment guidelines for relapsed
patients are difficult to establish. Conventional therapies with
ATRA and CHT ± BMT remain the most sound treatments
although even in this context data are restricted to small series
treated heterogeneously.1–5,56,57,129 In most institutions,
aggressive consolidation followed by BMT would still be used,
after ATRA-induced second CR, as a first option for relapsed
APL. It is conceivable that rapid generation of data on the use
of the above-mentioned novel compounds will change our
routine approach to relapsed APL in the near future. Regardless of the therapeutic choice, the achievement of a stable
second molecular CR should remain our therapeutic goal.
Minimal residual disease (MRD) and molecular relapse
Amongst all acute leukemias, APL is the only form in which
a specific disease marker, ie the PCR amplifiable PML/RAR␣
fusion gene, allows molecular assessment of response to treatment and MRD evaluation in 100% of patients. The prognostic value of detecting MRD during clinical remission was
initially suggested in Italy in 1992131 and successively confirmed in numerous studies conducted in China, USA, Japan
and several European countries.132–143 Studies performed by
using PCR assays capable of detecting one leukemic cell in a
background of 103–104 normal cells, agreed that a positive
PCR test at completion of consolidation is strongly associated
with impending clinical relapse. On the contrary, negative
tests are not always associated with prolonged DFS and cure.
In fact, a sizable proportion of patients who had tested negative post-consolidation were reported in the same studies to
have relapsed thereafter.131–143 Notwithstanding, it is worth
noting that no patients in long-term survival are found PCRpositive using this sensitivity threshold.131–143 This clearly indicates that a status of PML/RAR␣ PCR negativity is our best
presently available therapeutic goal.
A number of complex technical issues were extensively
debated which may account for difficulties in performing the
PML/RAR␣ RT-PCR test, and which contribute to explain its
relatively poor sensitivity.5,65,144 Usually, RNA yield is poorer
in APL as compared to other leukemias. In addition, low
efficiency of the reverse transcription step, coupled to instability of the fusion gene are further factors related to frequently
reported false negative results.5,65,144 Some recent studies have
outlined the possibility of improving the reaction sensitivity
and specificity. For example, RNA denaturation before the
reverse transcription step and/or use of the ‘hot start’ PCR
technique have been reported as useful means to decrease
the amplification of non-specific products and to allow more
specific primer annealing, thereby resulting in increased sensitivity.144,145 Furthermore, employment of the more sensitive
RAR␣/PML assay in conjunction with the conventional
PML/RAR␣ reciprocal assay may lead to the detection of MRD
in a greater number of patients, as discussed by D Grimwade
(London, UK).72,141 The major drawbacks related to the clinical use of this reciprocal assay are related to its lack of
expression at diagnosis in 20 to 30% of patients,146 and to
the occasional presence of residual transcripts in long-term
survivors.147 In the near future, a greater degree of inter-laboratory standardization should be made available through the
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F Lo Coco et al
input of automated, so-called ‘real-time’, quantitation of the
fusion transcript.
Recently, a large prospective study of the Italian GIMEMA
group confirmed the value of PCR positivity during remission
as a predictor of clinical relapse.148 In fact, of 21 patients who
converted to PCR-positive at any time during follow-up, 20
underwent hematologic relapse within a median time of 3
months, whereas only eight of 142 patients who were repeatedly PCR-negative post-consolidation relapsed during followup. These data were obtained on a longitudinal prospective
evaluation designed in 1993 in the context of the clinical
GIMEMA multicenter trial, with pre-established time intervals
for BM sampling, and centralized molecular biology evaluation using a PCR method allowing a sensitivity of 10−4. The
results provide compelling evidence that MRD detection, at
this sensitivity level is prognostically relevant in APL.148 For
these reasons, the GIMEMA group recently amended the
ongoing trial by anticipating treatment of relapse at the time
of minimal disease recurrence. In this respect, a status of molecular relapse was defined as the conversion from PCR-negative to PCR-positive after consolidation, confirmed in two successive bone marrow samples taken 2–4 weeks apart, and in
the absence of morphologically detectable leukemic blasts in
the bone marrow or peripheral blood.148
F Lo Coco (Rome, Italy) presented preliminary data on treatment of molecular relapse. Reinduction therapy for these
patients included standard doses of oral ATRA for 30 days
followed by MTZ and Ara-C as consolidation and autologous
BMT. Despite being too early for any conclusive statement,
the study suggests some advantages in anticipating treatment
of relapse at the time of minimal disease recurrence.149 In fact,
reinduction was associated with virtually no toxicity and
could be administered on an outpatient basis. Moreover, in
the majority of cases a second molecular remission was achieved with ATRA alone prior to chemotherapy consolidation,
a result that is only sporadically obtained when ATRA is
administered for overt relapse.62,149 Whether early therapeutic
intervention on MRD will improve the overall survival of APL
patients remains to be established on a longer follow-up.
Conclusions and future perspectives
A rather unusual simultaneity of important discoveries have
paved the way, in the early 1990s, to the impressive improvement in prognostic outcome of APL patients obtained in recent
years. Initial observations on the striking effect of differentiation therapy in this leukemia subset were immediately followed by identification of a disease-specific and pathogenically relevant gene abnormality. This concomitance,
which soon translated into important insights in APL development and allowed the design of more rationale and diseasetargeted therapy, turned out to be essential for converting this
once rapidly fatal malignancy into one of the most curable
human leukemias. Indeed, survival curves of APL patients
presented in this symposium are almost superimposable to
those seen in childhood ALL.
For a number of reasons, APL represents a paradigm for the
understanding of human leukemogenesis and for potential
development of innovative therapeutic strategies. As discussed
by TA Lister (London, UK) in his concluding remarks, areas of
special interest include: (1) the complex mechanisms involved
in leukemia-associated differentiation block and means to
release the latter; (2) the existence of discrete genetico-clinical
entities which are precisely defined at the molecular level and
require targeted treatment; and finally (3) the possibility of
improving survival by anticipating therapy of relapse in
patients showing disease recurrence at the molecular level.
In spite of recent progress, APL remains nowadays a curable, yet not cured disease. Several major clinical problems
still account for treatment failure, including early death and
disease relapse. In addition, patient subsets that may benefit
from more intensive therapy after remission induction have
not yet been identified. The promising therapeutic potential
of As03 and of other newly described agents still has to be
validated by testing their efficacy at various time-points and
in distinct combinations, including or not more conventional
agents known to be active in the disease, such as retinoids
and anthracyclines. Amongst other advances, early recognition and treatment of APL recurrence might represent a
further step towards the final cure of all patients affected by
this disease. As to this latter objective, we wish we could all
have the chance of celebrating its achievement in the near
future, hopefully during one of the forthcoming international
meetings on APL. No matter where this announcement will
be officially made, although no sites would appear to us more
appropriate than the Eternal City.
Acknowledgements
This work was supported by AIL (Associazione Italian contro
le Leucemie). We are grateful to Drs WH Miller, MA Sanz
and A Falanga for helpful discussion and critical reading of
the manuscript.
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