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Blood, Vol. 94 No. 1 (July 1), 1999:
pp. 12-22
REVIEW ARTICLE
Genetic Diagnosis and Molecular Monitoring in the Management of Acute
Promyelocytic Leukemia
By
Francesco Lo Coco,
Daniela Diverio,
Brunangelo Falini,
Andrea Biondi,
Clara Nervi, and
Pier Giuseppe Pelicci
From the Department of Cellular Biotechnology and Hematology, the
Department of Histology and Medical Embryology, University La Sapienza
of Roma; Clinica Pediatrica, University of Milano, Monza; Institute of
Hematology, University of Perugia; and the Department of Experimental
Oncology, European Institute of Oncology, Milano, Italy.
 |
INTRODUCTION |
DESPITE ITS well-known sensitivity to
anthracyclines,1-5 acute promyelocytic leukemia (APL) was
catalogued until the late 1980s as one of the most rapidly fatal human
tumors, with the majority of patients dying early of intractable
hemorrhage or disease relapse.6 Two major research
advances the cloning of the promyelocytic leukemia/retinoic acid
receptor  (PML/RAR) fusion gene underlying the t(15;17) specific
aberration7-9 and the advent of differentiation therapy
with all-trans retinoic acid (ATRA)10-12paved
the way in the early 1990s for the design of modern diagnostic
strategies and tailored treatment of the disease. Together
with a better control of the coagulopathy, these advances contributed
in the following years to a remarkable improvement in patients'
outcome.13-27 Recent data from large clinical trials conducted in Europe,19-22 the United
States,23-25 Japan,26 and China27
show that front-line treatments combining ATRA and
anthracycline-containing chemotherapy result in long-term survival and
potential cure in nearly 70% of patients. In addition to fostering
basic and clinical investigation on differentiation therapy, APL may
serve today as a paradigm for the development of tailored, genetically
targeted treatment of human leukemia.
Among hematologic malignancies, APL is one of the best-characterized
genetic-clinical entities. In fact, (1) a unique molecular lesion (the
PML/RAR fusion gene) segregates with a single phenotype (hyper- or
micro-granular French-American-British [FAB] M3 acute myeloid
leukemia [AML]) being only sporadically reported outside this
subset13-18; (2) the effectiveness of a specific therapy
(ATRA) is strictly conditioned by the presence of this genetic
abnormality in leukemic cells, and its detection identifies virtually
100% of ATRA-responsive patients.19,21-23,28 Besides
highlighting the pathogenetic role of PML/RAR, recent studies have
shown that the same hybrid protein is targeted by
ATRA.29-40 In view of these findings, and for the purpose
of correct therapeutic intervention, definition of APL requires
diagnostic identification of the specific genetic lesion or its protein
product.18
Together with conventional karyotyping, reverse
transcriptase-polymerase chain reaction (RT-PCR) for PML/RAR is
currently the most widely used method for the genetic diagnosis of
APL.18,41 Other recently developed and equally specific
techniques include fluorescence in situ hybridization (FISH) analysis
and PML immunostaining with specific antibodies.42-44
Compared with the latter, RT-PCR offers the additional advantage of
defining the PML/RAR isoform type and enables sensitive detection of
residual disease (MRD) during treatment and
follow-up.28,41,45-51 In some instances, the information
derived from prospective MRD monitoring of APL has already been
translated into operational therapeutic decisions, such as the
institution of salvage treatment at the time of PCR-detected minimal
disease recurrence.52-54 Nonetheless, this technique is not
devoid of drawbacks and difficulties, requiring skillful expertise as
well as appropriate inter-laboratory standardization for correct interpretation of results, especially in the setting of multicenter clinical trials.
After a brief update on the role of PML/RAR in APL pathogenesis and
response to ATRA, we discuss here the advantages and pitfalls of the
main procedures used for genetic diagnosis in this disease, with
special emphasis on RT-PCR amplification of the fusion gene. Further,
we review the clinical significance of PML/RAR isoforms and the
utility of RT-PCR studies to assess response to therapy and MRD monitoring.
| |
MOLECULAR PATHOGENESIS AND RESPONSE TO ATRA IN APL |
The APL phenotype is associated with chromosomal translocations
disrupting the RAR locus and resulting in fusion products with other
nuclear proteins (PML/RAR, PLZF/RAR, NuMA/RAR, or NPM/RAR13-18).
Despite clinical similarity, ATRA induces differentiation of leukemic
blasts and disease remission only in PML/RAR APLs, whereas
PLZF/RAR APLs are ATRA resistant.13-18 RAR-fusion
proteins interfere with the program of terminal differentiation and are probably involved in both disease pathogenesis and response to ATRA.13-18 In particular, (1) in vitro, ectopic expression
of PML/RAR, but not that of PLZF/RAR, increases ATRA-sensitivity
of hematopoietic cell lines and restores ATRA-sensitivity of resistant
cells29,55,56; (2) in vivo, PML/RAR and PLZF/RAR
transgenic mice develop leukemias, but only leukemias from PML/RAR
mice are ATRA-sensitive.30-33,40
Degradation of PML/RAR by proteaosomal pathways and activated
caspases has been proposed as a critical mechanism for ATRA-response of
APL blasts.34,35 However, inhibition of the PML/RAR
proteolysis does not impair ATRA-dependent differentiation, further
indicating a direct role for PML/RAR in this event.36
PML/RAR and PLZF/RAR retain the ability of RAR to regulate
transcription of ATRA-target genes and to recruit the
N-CoR/histone-deacetylase (HD) complex, which lead to a repressive
chromatin conformation.37-40,57-59 High doses of ATRA
release HD activity from PML/RAR but not from PLZF/RAR, which
contains a second N-CoR/HD binding site in the PLZF moiety. Mutation of
the N-CoR binding site abrogates the ability of PML/RAR to block
differentiation, whereas inhibition of HD activity switches PLZF/RAR
from an inhibitor to an activator of the ATRA-signaling
pathway.38 Therefore, recruitment of the N-CoR/HD and
regulation of ATRA-target genes are crucial to the transforming
potential of RAR-fusion proteins.
RAR fusion proteins' biological activity may also interfere with
cell survival, and this may contribute to their leukemogenic potential.
Expression of PML/RAR in hematopoietic cells inhibits programmed
cell death, whereas in nonhematopoietic cells it induces apoptosis.29,60,61 Indeed, forced expression of PML in a
variety of cell lines induces growth arrest through apoptotic
pathways62-65 while targeted disruption of the PML locus in
mice increases the rate of spontaneous or induced
carcinogenesis.66 The molecular mechanisms by which
PML/RAR deregulates PML intracellular pathways are not clear. PML
localizes within distinct nuclear compartments (nuclear bodies;
NB).67-69 The PML-NB structure is destroyed in APL cells
and is restored after ATRA treatment, as a direct consequence of
PML/RAR degradation.36 Also, PLZF is a growth suppressor that localizes within nuclear bodies.70 Thus, nuclear
bodies could be involved in negative growth control and their
deregulation by RAR-fusion proteins might represent a critical step
during promyelocytic leukemogenesis.
| |
TECHNICAL APPROACHES FOR GENETIC DIAGNOSIS OF APL |
Compared with diagnosis of other AML subtypes, the identification of
APL conveys unique therapeutic and prognostic implications. In fact,
(1) this leukemia is a medical emergency and up to 10% of early
hemorrhagic deaths are currently recorded even in patients receiving
modern state-of-the-art treatments19-27; (2) the optimized front-line approach (ATRA + chemotherapy) is different from that used
in other AMLs, and is effective also in controlling the
life-threatening coagulopathy.13-18 Although morphologic
diagnosis is straightforward in the vast majority of hypergranular
cases, it appears insufficient for the identification of each and every
patient who would benefit from ATRA-containing treatments. In fact,
ATRA is effective in leukemia cells expressing the PML/RAR protein,
whereas cases with cytological features of APL but bearing variant
translocations such as the t(11;17) expressing the PLZF/RAR fusion
are ATRA-unresponsive.13-18,71 Vice versa, the APL
microgranular variant, which expresses PML-RAR and is
ATRA-sensitive,13-18 is, on morphological grounds, hardly distinguishable from AMLs with differentiation (M2) or from
myelomonocytic leukemias (M4).72 The APL-specific genetic
abnormality can be demonstrated at the chromosome, DNA, RNA, or protein
level. We review here the main advantages and pitfalls of these
methods, with special emphasis on RT-PCR of PML/RAR.
Cytogenetics and Southern blot.
Conventional karyotyping on banded metaphases enables documentation of
the pathognomonic t(15;17) in the majority (up to 90%) of
morphologically defined APL cases.13-18 A recent study of
patients entered into the Medical Research Council (MRC) ATRA trial
showed that the translocation could be cytogenetically documented in 81 of 93 (87%) cases with PCR-detectable PML/RAR
transcript.42 Reasons for "false-negative"
cytogenetics in APL include analysis of cells not belonging to the
neoplastic clone (eg, erythroblasts), which may undergo mitosis in
direct or short-term preparations,73 the existence of
cryptic translocations or microinsertion,74,75 or
difficulties in interpreting poor-quality preparations with few mitoses
and/or fuzzy chromosomes.42 In most of these situations, FISH analysis using specific PML and RAR probes would successfully assist diagnosis and demonstrate the genetic
rearrangement.74,75 Despite these limitations, conventional
karyotype retains an important role and should always complement, and
not be a substitute for, molecular diagnostics. In fact,
chromosome partners of 17q other than 15q,76-78 complex
translocations involving more than two chromosomes,79,80 and additional abnormalities besides the t(15;17)81-83
are only detectable by cytogenetic studies. Their identification
provides potentially relevant clinical and biological information. Of
considerable interest, in view of the different therapeutic
requirements, is the identification of the rare t(11;17) APL
variant.71
Southern blot is equally as specific as RT-PCR but laborious and
time-consuming (5 to 10 days). In fact, at least two probes and several
enzymatic digestions are required to identify breakpoints on the RAR
second intron in all APL cases.84,85 Additional hybridizations with PML probes are needed to detect rearrangements on
the 15q+ derivative, to determine the type of PML breakpoint, or to
rule out a variant translocation.86 Because of such
complexity, the use of Southern blot for routine diagnosis of APL has
been almost abandoned in specialized laboratories.
Staining with anti-PML antibodies.
The study of the PML distribution pattern in leukemic cells provides a
rapid, specific, low-cost, and relatively simple diagnostic approach.42-44 Different from the wild-type (speckled)
staining, which corresponds to the localization of PML into 5 to 20 discrete nuclear particles (so-called "nuclear bodies"), APL
cells show a characteristic and easily distinguishable nuclear PML
positivity known as "microspeckled," resulting from the
disruption of the nuclear bodies and redistribution of the protein into
greater than 50 small granules/per cell (Fig
1).42-44 Either
immunocytochemistry or immunofluorescence have been successfully used
as detection systems. The monoclonal antibody (MoAb)
PG-M3,87 directed against an amino-terminal PML epitope
that is shared by all the known PML and PML-RAR isoforms, is
particularly suitable for diagnostic use.44 In view of its
reliability for rapid (2 hours required) and specific diagnosis,
immunocytochemistry might be recommended as a valid alternative to
molecular or karyotypic analysis.
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| Fig 1.
Immunolabeling of a microgranular APL case (A) and of an
acute nonlymphoid leukemia M4 subtype (B) with the PG-M3 MoAb. (A)
Indirect immunofluorescence with rhodamine-labeled antibody and nuclear
DAPI counterstain. Double arrows show a microspeckled distribution of
the PML/RAR protein within the nuclei of APL cells. This pattern
contrasts with the nuclear speckled positivity PML wild type of normal
residual hematopoietic elements (single arrow). (B) Indirect
immunofluorescence with fluoresceine-labeled antibody and nuclear DAPI
counterstain. Blast cells show the nuclear speckled positivity.
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RT-PCR of PML/RAR.
This is the only technique that defines the PML breakpoint type and
that allows the definition of a correct strategy for subsequent MRD
monitoring. The advantage of routinely using this assay at diagnosis to
better address treatment was initially suggested by Miller et
al,28 and subsequently validated in prospective multicenter
trials published by the Gruppo Italiano Malattie Ematologiche Maligne
dell'Adulto (GIMEMA),19 Medical Research Council
(MRC),21 and Programa Español para el Tratamiento de
Hemopatias Malignas (PETHEMA)22 groups.
According to most investigators, high-quality RNA and efficient RT are
the crucial determinants for successful RT-PCR of
PML/RAR.88-90 Because of frequent leukopenia and the associated coagulopathy, the yield and quality of RNA from diagnostic samples are frequently poor. It is the experience of the GIMEMA study,
now including almost 700 newly diagnosed APL patients, that local
preparation of mononuclear cells (Ficoll-Hypaque [Nycomed Pharma AS,
Oslo, Norway] isolation and storage in a guanidium-isothiocyanate [GTC] solution) and overnight delivery to a referral laboratory results in better RNA quality, compared with shipment of whole blood.19 For the purpose of rapid diagnosis,
small volumes (0.5 mL) of GTC-suspended leukemic cells can be processed
with bench microfuges, thereby avoiding the use of expensive equipment
and reducing RNA extraction times to less than 1 hour. After RT, a hot-start PCR may be performed at diagnosis with a single amplification round. This procedure allows visualization of the results on the ethidium bromide-stained gel in 3 to 4 hours (including time required for RT).90 By avoiding or minimizing primer misannealing
and dimerization, the hot-start method enables a more specific reaction that also results in enhanced sensitivity. Denaturation of RNA before
RT and initial denaturation of first-round PCR products have also
been recommended by some investigators for improved specificity
and sensitivity of the assay.89 Blotting of PCR products
and hybridization with specific probes is required if the amplification
of nonspecific bands is suspected. This is particularly important in
light of the frequent occurrence of PCR artifacts resulting from primer
misannealing. As a control for RNA integrity and efficiency of the RT
step, some investigators amplify one of the two normal genes involved
in the translocation, whereas others prefer the analysis of an
unrelated, ubiquitously and low expressed gene.91
In the clinical practice, patients with typical hypergranular APL are
usually given specific ATRA-containing therapy before results of the
genetic characterization are known. This attitude carries
the sole risk of initiating ATRA in those rare cases with hypergranular
morphology that lack the PML/RAR recombination. Given the
possibility of rapidly identifying APL at the genetic level by anti-PML
staining, an anti-PML specific antibody could be included in the
characterization panel routinely used in all AMLs to minimize the risk
of mistreating patients. This would also avoid the risk of missing
those APLs with atypical morphology with no available cytogenetic and
molecular characterization.
| |
CLINICAL SIGNIFICANCE OF THE DISTINCT PML/RAR ISOFORMS |
Because of chromosome 15 breakpoint variability (PML intron 6, bcr1;
exon 6, bcr2; intron 3, bcr3), two transcripts of different length can
be visualized by RT-PCR, referred to as long (L) and short (S). L
includes bcr1 and bcr2 or variable (V) transcripts (Fig
2), which can only be distinguished by DNA
sequence or specific oligonucleotide hybridization. Together, bcr1 and
bcr3 transcripts account for more than 90% of cases. Correlations
between type of PML/RAR transcript and patients' clinico-biologic
features at diagnosis or response to therapy and outcome have been
investigated in many studies.19,42,45,50,82,92-101 The
majority of such studies, however, used methodologies that fail to
distinguish the L from the V form of PML/RAR; therefore, the impact
of including V form patients in the L form group is unknown.
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| Fig 2.
Schematic representation of the three major PML/RAR
fusion transcripts. Breakpoints on the RAR gene always occur within
intron 2. Due to distinct breakpoints on the PML gene (intron 6, exon
6, and intron 3), different segments of this latter are fused to RAR
exon 3, resulting in bcr1 (L type), bcr2 (V type), and bcr3 (S type)
fusion transcripts, respectively. Numbers in boxes represent PML and
RAR exons.
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With respect to presenting features, no correlations were found with
sex, platelet number, presence of the coagulopathy, and ATRA
syndrome.19,45,97,99 The bcr3 transcript was reported to
correlate with higher white blood cell counts and M3v
morphology,99 additional karyotype
abnormalities,82 CD34 expression,92 and CD2
expression.92,93 Association with additional
karyotype abnormalities and positive staining for CD2 was, however, not confirmed by others.42,94,95 The distribution of PML/RAR isoforms according to age showed a higher frequency of the bcr3 type in
children with respect to adults in the Italian
population,92 whereas Kane et al100 reported
that the V form was the most frequent in a series of the US Pediatric
Oncology Group.
The results of studies that analyzed response to treatment and clinical
outcome according to the PML/RAR isoform need to be carefully
evaluated, taking into consideration relevant differences in the
therapeutic context. Shorter remission duration in patients with the
bcr3 isoform were reported in two earlier studies in which ATRA alone
was used as induction therapy.50,96 In line with these
findings, in vitro studies have shown significant differences in
retinoid binding and transcriptional activation properties of bcr1 and
bcr3 PML/RAR isoforms.102 Subsequent surveys of patients
receiving combined ATRA and chemotherapy (in most cases administered
simultaneously) showed no statistically significant differences in
complete remission and disease-free survival
rates.19,99,101 However, it is worth noting that a trend
toward less favorable prognosis is reported in all studies for patients
with the bcr3 isoform.19,99,101 By analyzing molecular
response to treatment in the GIMEMA protocol, we found no difference in
breakpoint distribution between patients achieving RT-PCR negativity
and those remaining positive after induction.19 Finally, a
study of Gallagher et al98 reported a decreased response to
ATRA in vitro in patients with a bcr2 (V) PML/RAR transcript.
Taken together, these data indicate that, although a bcr3 transcript
type correlates with established adverse prognostic features in APL
(ie, hyperleucocytosis, M3v), such association does not appear, at the
present time, to translate into poorer outcome, at least in the context
of modern ATRA + chemotherapy regimens. However, a long-term follow-up
of ongoing clinical trials is awaited to better clarify this issue.
| |
MOLECULAR RESPONSE TO TREATMENT AND MRD MONITORING |
The presence of a specific and PCR-detectable tumor marker in leukemic
cells allows investigators to molecularly assess response to therapy
and MRD in every patient with APL. Many investigators have monitored
MRD by RT-PCR during hematologic remission to better adjust
treatment.18,103 Overall, there is general agreement that a
positive PML/RAR test after consolidation is a strong predictor of
subsequent hematologic relapse, whereas repeatedly negative results are
associated with long-term survival in the majority of patients (Table
1).28,45,52,88,104-113 However, these correlations are not absolute. In fact, either sporadic cases who
remain PCR+ in long-term remission114 or, more
frequently, patients who ultimately relapse after negative
tests,45,52,88,103,107-111 have been reported. Several
reasons may account for such exceptions. Most of initial studies were
retrospective and heterogeneous in terms of patient selection,
treatment, and sensitivity of the PCR assays used.28,45-50
In addition, one major limitation of the assays used is their failure
to precisely quantitate the amount of MRD, which in turn makes a
comparison among the reported studies difficult. Recent prospective
analyses on homogeneously treated patients, and with more rigorous
methodologies, have further highlighted the prognostic value of RT-PCR
monitoring in APL (Table 1).19,52,88,101,107-110 In this
section, we will critically review the studies reported on this topic,
with special emphasis on the findings of most recent large, prospective
trials.
The following parameters are critical for the interpretation of MRD
studies: (1) timing of sampling; (2) therapeutic context; (3) fusion
gene (PML/RAR or RAR/PML reciprocal hybrid) and control gene of
the PCR (actin,  2 microglobulin, PML, RAR, others); (4)
sensitivity of the RT-PCR assay; (5) inter-laboratory standardization (particularly relevant in multicenter studies); (6) retrospective or
prospective nature of the study; (7) number of tests per each patient.
With concern to technical aspects, the recommendations previously
discussed for diagnostic RT-PCR are valid and even more important in
the setting of MRD studies. False-negative results are a major clinical
concern and may result from poor RNA quality or limited sensitivity of
the assays used. However, low RT-PCR sensitivity may also depend on
properties of the PML/RAR transcript, such as low expression or
relative instability.18,41,89 To increase sensitivity of
the assay, a number of technical modifications have been suggested
(Table 2).18,89,90 As internal
PCR controls, dilutions of low-expressed genes (devoid of pseudogenes
to rule out amplification of DNA) should be used to mimic the amounts of MRD searched for the specific translocation.91 Finally,
continuous efforts should be done toward standardization of the
PML/RAR RT-PCR assay to guarantee reproducibility of results among
different laboratories. A certain degree of inter-laboratory
discordance was documented by a recent external quality-control study
involving 12 European institutions.115 The use of the newly
developed real-time PCR technology may hold promise to provide adequate
standardization at the quantitative level and more objective comparison
of results.
With respect to timing of sampling, no differences in the 2-year
disease-free survival were found in 139 patients of the GIMEMA trial
who tested positive or negative
postinduction.19 Similarly, the MRC reported
no statistically significant differences in the 3-year relapse risk
according to PCR status at the end of induction treatment.101 It is hoped that such early monitoring using
quantitative techniques would result in more informative evaluations,
thereby allowing investigators to better address further therapy in
these patients.
As to the tests performed during consolidation, detection of residual
disease after the third course (of four total given) correlated in the
MRC study with an increased risk of relapse (62 v 29%,
P < .01) and with poorer survival.101 RT-PCR
tests during consolidation were performed in too few cases in our
study,19 and no conclusion on their utility can be drawn at
this time. In both the GIMEMA and MRC trials, patients were studied
prospectively and with a PML/RAR RT-PCR assay showing a similar
sensitivity threshold (1 positive cell in 104). In the
MRC analysis, patients were additionally evaluated for the reciprocal
RAR/PML hybrid amplification, which as also found by Tobal et
al114 is reported to be more sensitive, with a
detection limit of 1 in 105 or even greater. Although this
approach did increase the detection rate of residual disease during
consolidation, its use failed to identify all patients who ultimately
relapsed.101 Furthermore, because RAR/PML is not
expressed in a quarter of patients at diagnosis116 and
because of occasional detection of residual RAR/PML transcripts in
some long-term survivors, its role in molecular monitoring is uncertain
at present.
All MRD studies conducted in patients receiving chemotherapy in
addition to ATRA reported an exceedingly high proportion (>90%) of
RT-PCR cases at the end of
consolidation,19,21,52,101 implying that MRD evaluation at
this sampling time fails to identify the majority of patients that will
eventually relapse ( 25% to 30%).19,21,52,101 Increased
sensitivity of the PML/RAR assay might lead to better identification
of this sizable fraction of patients who require additional therapy at
the end of consolidation. However, improved sensitivity might carry the
risk of detecting "indolent" transcript amounts, such as those
occasionally reported in long-term survivors.18,114
Although assessment of the PCR status immediately after the end of
consolidation has little prognostic role, subsequent monitoring during
the early posttreatment period appears informative. We recently
reported the results of a prospective study in which 163 patients of
the GIMEMA trial were tested at regular preestablished time intervals
after the end of treatment.52 Twenty of 21 patients who
converted to PCR+ relapsed within a median time of 3 months, whereas the 3-year estimate of relapse risk for patients who
tested negative more than two times postconsolidation was below 10%.
In fact, only 8 of 142 who tested negative in at least two successive
samples underwent relapse after a median follow-up of 18 months
postconsolidation. Interestingly, 81% of the PCR+
conversions were recorded within the first 6 months after the end of
therapy.52 It is based on these findings that, in our trial, patients in clinical remission who convert to
RT-PCR+, as confirmed in two successive BM samplings, are
now administered early salvage treatment, before developing overt
relapse.53
In summary, RT-PCR of PML/RAR appears less sensitive than the assays
used to amplify other leukemia-associated hybrid genes; however, the
assessment of remission status at the molecular level represents a
significant clinical advance with respect to other poorly sensitive
methods (morphology, karyotype). It is commonly accepted that the
efficacy of present strategies or novel approaches for the treatment of
this disease (arsenicals, liposomal ATRA, other retinoid derivatives,
etc) need to be assessed taking into account the response at the RT-PCR
level. However, repeated posttreatment sampling appears necessary to
obtain prognostically relevant information. A status of persistent
PCR-negativity confirmed in at least two successive marrow samples at
the end of treatment might be regarded as our best presently available
therapeutic goal. While further standardization of laboratory tests and
automated quantitation (real-time PCR) will probably improve the
current state-of-the-art and allow us to better identify distinct risk
groups at the end of treatment, prospective monitoring during the first
6 to 12 months is recommended for early detection of relapse.
| |
CONCLUSIONS AND FUTURE PERSPECTIVES |
Elucidation of the genetic defect underlying the disease-specific
t(15;17) has had a remarkable impact into the management of APL.
However, while the relevance of diagnostic PML/RAR detection is out
of question, further investigation is needed to better establish the
clinical value of molecular monitoring. In particular, the following
have to be clarified: (1) whether a greater sensitivity of the RT-PCR
assay for PML/RAR will allow better identification of patients at
risk of relapse at the end of consolidation; (2) if newer quantitative
technologies may provide earlier monitoring that is clinically
significant; and (3) whether anticipation of salvage therapy in
patients treated for molecular recurrence is advantageous over the
treatment of hematologic relapse. As to this latter issue, studies are
underway,53 but it may be anticipated that therapy of
molecular relapse should at least minimize the significant mortality
rate observed during reinduction of overt disease.
The success of tailored treatment of APL encourages both basic and
clinical investigators to search for more rational, and, hence, less
toxic drugs that directly interfere with the function of the
leukemia-associated fusion proteins (molecular
treatment). Like PML/RAR and PLZF/RAR, AML1/ETO,
the transforming protein associated with M2-AMLs, has been recently
shown to function as a transcriptional repressor in a complex
containing nuclear corepressors and
histone-deacetylases.117,118 Also in this case, the nuclear complex formation is crucial to the biological activities of the fusion
protein. Translocations involving the histone-deacetylases p300 and CBP
have been recently described in rare cases of AML.119 Thus,
formation of aberrant complexes with histone-modifying enzymes and
alterations in chromatin structure might be a general mechanism of
myeloid leukemogenesis. In vitro, drugs with inhibitory activity on
histone-deacetylases have already been shown to interfere with the APL
phenotype.38-40 It is tempting to speculate that the use in
AMLs of drugs that modulate histone acetylation might, in the near
future, represent another important example of molecular treatment.
| |
ACKNOWLEDGMENT |
We are grateful to Profs F. Mandelli and M.A. Sanz, and to Drs D. Grimwade, G. Avvisati, W. Arcese, and G. Cimino for helpful discussion
and critical reading of the manuscript. We also thank Prof A.K. Burnett
and Dr D. Grimwade for sharing their unpublished data. Finally, we are
indebted to all the clinical and laboratory investigators of the GIMEMA
and Associazione Italiana di Ematologia e Oncologia Pediatrica (AIEOP)
groups for their invaluable contribution to the Italian cooperative
study "AIDA" for the diagnosis and treatment of patients with
acute promyelocytic leukemia.
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FOOTNOTES |
Submitted October 19, 1998; accepted April 9, 1999.
Supported by AIL (Associazione Italiana contro le Leucemie), AIRC
(Associazione Italiana per la Ricerca sul Cancro), CNR P.F. "Biotecnologie," Italia-Usa Project on Therapy of tumors, and Fondazione Tettamanti.
Address reprint requests to Francesco Lo Coco, MD, Department of
Cellular Biotechnology and Hematology, University La Sapienza of Roma,
Via Benevento 6, 00161 Roma, Italy; e-mail:
lococo{at}bce.med.uniroma1.it.
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INTRODUCTION
MOLECULAR PATHOGENESIS AND...