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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 92 No. 4 (August 15), 1998:
pp. 1172-1183
Leukemic Cellular Retinoic Acid Resistance and Missense Mutations
in the PML-RAR Fusion Gene After Relapse of Acute Promyelocytic
Leukemia From Treatment With All-trans Retinoic Acid and
Intensive Chemotherapy
By
Wei Ding,
Yun-Ping Li,
Lucio M. Nobile,
George Grills,
Ines Carrera,
Elisabeth Paietta,
Martin S. Tallman,
Peter H. Wiernik, and
Robert E. Gallagher
From the Montefiore Medical Center and Albert Einstein Cancer Center,
Bronx, NY; the Northwestern University Medical School, Chicago, IL; and
the Eastern Cooperative Oncology Group, Brookline, MA.
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ABSTRACT |
This study evaluated whether relapse of acute promyelocytic leukemia
(APL) patients from clinical remissions achieved and/or maintained with all-trans retinoic acid (RA) in combination
with intensive chemotherapy is associated with leukemic cellular
resistance to RA and with alterations in the PML-RAR fusion gene. We
studied matched pretreatment and relapse specimens from 12 patients who received variable amounts of RA, primarily in nonconcurrent combination with daunorubicin and cytarabine (DA) on Eastern Cooperative Oncology Group (ECOG) protocol E2491, and from 8 patients who received DA only
on protocol E2491. Of 10 RA-treated patients evaluable for a change in
APL cell sensitivity to RA-induced differentiation in vitro, 8 showed
diminished sensitivity at relapse, whereas, of 6 evaluable patients
treated with DA alone, only 1 had marginally reduced sensitivity. From
analysis of sequences encoding the principal functional domains of the
PML and RAR portions of PML-RAR, we found missense mutations in
relapse specimens from 3 of 12 RA-treated patients and 0 of 8 DA-treated patients. All 3 mutations were located in the ligand binding
domain (LBD) of the RAR region of PML-RAR. Relative to normal
RAR1, the mutations were Leu290Val, Arg394Trp, and Met413Thr. All
pretreatment analyses were normal except for a C to T base change in
the 3 -untranslated (UT) region of 1 patient that was also
present after relapse from DA therapy. No mutations were detected in
the corresponding sequences of the normal RAR or PML (partial)
alleles. Minor additional PML-RAR isoforms encoding truncated PML
proteins were detected in 2 cases. We conclude that APL cellular
resistance occurs with high incidence after relapse from RA + DA
therapy administered in a nonconcurrent manner and that mutations in
the RAR region of the PML-RAR gene are present in and likely
mechanistically involved in RA resistance in a subset of these cases.
© 1998 by The American Society of Hematology.
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INTRODUCTION |
ALL-TRANS RETINOIC ACID (RA),
which functions by inducing terminal granulocytic differentiation of
acute promyelocytic leukemia (APL) cells,1-4 is a highly
effective agent for inducing complete hematologic and clinical
remission (CR) in APL patients.5-13 Variations in the
reported incidence of RA-induced CR in APL patients (72% to 100%)
have been related to early disease complications, to RA toxicity, or to
other extenuating circumstances (such as inadequate diagnostic
criteria).5-12 We are not aware of any well-documented case
of primary clinical resistance to RA in a previously untreated APL case
determined either to have the classical 15;17 chromosome translocation
and/or to express the pathognomonic PML-RAR fusion gene
product. On the other hand, acquired clinical resistance universally
develops within a few months in APL cases treated with RA as a single
agent.5-7,9 This universality logically suggests some
regulatory systemic mechanism of acquired resistance.14 This hypothesis has been supported by the finding that there is a rapid
decrease in achievable plasma RA levels within a few days of beginning
RA therapy.15-17 This, in turn, has been related to the
induction of cytochrome P-450 enzymes and lipid peroxidases that
enhance the metabolism of RA15,16,18 and to the induction
of a cytoplasmic RA binding activity, cellular retinoic acid binding
protein-II (CRABP-II), in various types of cells, including
skin17 and bone marrow cells,19,20 that may
then act as a systemic sump to diminish the amount of RA able to reach
crucial molecular response sites in the nucleus of APL cells.4,14 However, some observations suggest that these
systemic considerations may not be a sufficient explanation for the
development of clinical ATRA resistance. Of particular note are
observations that APL cells from many patients who relapse after RA
therapy have reduced sensitivity to RA-induced differentiation in
vitro,6,20,21 suggesting the presence of significant
intrinsic APL cellular RA resistance. Most of these reported cases were
in second relapse, having previously relapsed from chemotherapy-induced
remissions, and many were treated with various types of low-dose
consolidation or maintenance chemotherapy. Thus, it is of interest to
determine the incidence of leukemic cellular resistance in previously
untreated APL cases who relapse from remissions achieved on RA in
combination with intensive consolidation chemotherapy, which has been
demonstrated to be necessary for high long-term disease-free survival
rates.4,8,12
Two molecular alterations have previously been associated with the
development of intrinsic APL cellular RA resistance. First, CRABP-II
protein RA binding activity was reported to be markedly increased in
fresh bone marrow specimens from 4 RA-treated APL patients after
clinical relapse compared with absent levels in pretreatment
specimens.20 From these observations, it was suggested that
cytoplasmic sequestration and catabolism of RA might produce intrinsic
APL cellular RA resistance by impeding RA nuclear
access.4,20 However, we have consistently found
constitutive expression of CRABP-II mRNA and of cytoplasmic RA protein
binding activity in the leukemic cells of previously untreated APL
cases (Hallam et al22 and Zhou et al, manuscript
submitted), raising uncertainty about the general
importance of this proposed mechanism. Second, in the established APL
cell culture line NB4, abnormalities of RA binding and of nuclear
interprotein complex formation by the APL-specific PML-RAR fusion
gene product have been reported in several sublines selected for RA
resistance in vitro.23-27 In two sublines, these were
related to missense mutations in the ligand binding domain (LBD) of the
RAR region of PML-RAR.25,28 Notably, mutations in the
LBD of normal RAR have also been related to the development of RA
resistance in independently selected sublines of the RA-sensitive
myeloid leukemia cell line HL-60.29-31 However, in the only
reported analysis of fresh APL specimens, no mutations were found in
the LBD of RAR in either the chimeric or normal allele in 20 APL
cases, including 5 cases with acquired RA resistance.32
In the current study, we tested matched pretreatment and relapse APL
cells from 19 patients treated on Eastern Cooperative Oncology Group
(ECOG) protocol E2491 with RA + chemotherapy (daunorubicin and
cytarabine [DA]) or, as controls, with DA alone12 for in vitro sensitivity to RA-induced differentiation and for gross or fine
abnormalities of PML-RAR mRNA. In addition, we made this assessment
in 1 protocol patient and 1 non-protocol patient who were further
treated with intravenous liposomal RA after relapse from oral
RA-containing therapy. The scope of the analysis was affected not only
by the possibility of finding LBD mutations in RAR sequences in a
manner analogous to those in resistant sublines of HL-60 or NB4, but
also in consideration of reports that the PML portion of PML-RAR is
importantly involved in the APL cell response to RA33-37
and of a report that the RING finger and second B-box domains of the
PML-region of PML-RAR were consistently mutated in a
PML-RAR-induced avian leukemia model.38 The results of
our analysis indicated that APL cellular RA resistance occurs with high
incidence in patients relapsing from nonconcurrently administered RA + DA and that in a subgroup of these patients this is likely due to
missense mutations in the RAR region of PML-RAR. These are the
first reported mutations of PML-RAR under clinical conditions and, more generally, are the first reported mutations of RAR sequences in
naturally occurring tumor cells in vivo. Identification of these
mutations in only a subgroup of patients with APL cellular RA
resistance also indicates that the molecular mechanisms of resistance
are heterogeneous.
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MATERIALS AND METHODS |
Patients and pretreatment clinical laboratory studies.
Nineteen of the 20 patients involved in this study were participants in
ECOG clinical trial E2491, which is a subset of previously untreated
adult APL patients studied on intergroup protocol INT 0129.12 One patient was a non-protocol patient from the
Montefiore Medical Center Oncology Service. Patient materials were
obtained under an ECOG protocol-approved consent form and/or
individual institutional review board approval. Baseline patient data,
eg, white blood cell (WBC) count, for ECOG cases were obtained from the
central data registry.
Cell preparation and in vitro tests for RA sensitivity.
Heparinized bone marrow (BM) and peripheral blood (PB) specimens were
received by overnight express mail from ECOG institutions at room
temperature. A low-density WBC fraction (density  1.077 g/mL) was
isolated and evaluated as previously described.39 Isolates
with  75% myeloblasts/promyelocytes were used for the assesment of in
vitro APL cell sensitivity to RA, as previously described.30,39
Reverse transcription-polymerase chain reaction
(RT-PCR) and allele-specific nested PCR analysis.
Total cellular RNA was prepared by a modified protocol of the guanidine
isothiocyanate extraction-cesium chloride gradient ultracentrifugation
procedure, as previously described,30 or using the TRIzol
reagent (Life Technologies, Inc, Gaithersburg, MD), according to the
supplier's instructions. Reverse transcription of RNA from random
hexamer primers (Pharmacia, Piscataway, NJ) and PCRs were performed
essentially as previously described,39 except that
amplification conditions were modified to be optimal for each primer
pair and 2% formamide was included to reduce nonspecific signals.40 Primer pairs were designed using Oligo software
(National Biosciences, Inc, Plymouth, MN) and synthesized by Perkin
Elmer (Norwalk, CT) via the Albert Einstein College of Medicine.
For allele-specific nested PCR, an aliquot of first-round PCR product
was diluted (10- to 100-fold), and 1 to 2 µL was used as template for
the second-round PCR reaction following the same protocol as above. All
PCR products were analyzed through 3% agarose gels run in TAE (0.04 mol/L Tris-acetate, pH 7.2, 1 mmol/L disodium EDTA) buffer.
DNA sequence analysis.
PCR products were purified either with a QIAquick PCR
Purification Kit (QIAGEN, Hilden, Germany) or recovered from a
low-melting temperature agarose gel with the Wizard PCR Preps DNA
Purification System (Promega, Madison, WI). These DNA fragments were
directly cycle-sequenced with the corresponding PCR primers on an
automated DNA sequencer using dye terminators and AmpliTaq DNA
polymerase (Applied Biosystems, Foster City, CA). Sequencing results
were edited using the Gene Inspector software (Textco, West Lebanon, NH).
PML-RAR RT-PCR analysis.
To classify the PML-RAR mRNA type, primer pair I
(Fig 1) was used: IS (sense),
5-ACCGATGGCTTCGACGAGTTC-3; IAS (antisense), 5-AGCCCTTGCAGCCCTCACAG-3. For V-form PML-RAR cases,
nested PCR with primer pair II was applied and further verified
by sequence analysis, as described39: IIS,
5-CAATACAACGACAGCCCAGAA-3; IIAS(1),
5-TCACAGGCGCTGACCCCATA-3; IIAS(2),
5-GACCCCATAGTGGTAGCCTGAGGA-3.
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| Fig 1.
Schematic structure of PML-RAR mRNA showing the major
functional domains. The PML portion is hatched and the RAR portion is clear. Numbers in the boxes indicate exons. Vertical arrows indicate
the positions of break/fusion sites in the translocated PML gene that
result in the formation of three different forms of PML-RAR mRNA:
S-, V-, and L-forms. Arrowheads show the sites of PCR primers used in
this study. Primers used for screening of the PML region are shown
beneath the schematic bar, whereas those for the RAR region are
shown above it. The asterisks indicate the positions of base changes
found in the PML-RAR allele, and the coding region amino acid
changes are shown in expanded form below the schematic bar. The
abbreviations are as follows: Pro, proline-rich domain; Cys,
cysteine/histidine clusters; -Helix, -helical coiled-coil
dimerization domain; B-F, B- to F-region of RAR; ZF, zinc-finger
RING motif; BB1 and BB2, two B-boxes; DBD, DNA binding domain; DD,
dimerization domain; LBD, ligand binding domain; AF-2, activator
function-2 domain; AF-2 AD CORE, 7 amino acid long AF-2 activation
domain; 3-UT, 3 untranslated region.
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Strategy for PML sequencing.
PML-RAR allele-specific RT-PCR was performed first by primer pair P
(Fig 1): PS, 5-CCCAGCCCCAGCCCCAGCCCTACA-3; PAS,
5-AGCGGTTCCGGGTCACCTTGTTGA-3. For nested PCR, four sets
of inner primers (III through VI) were used for exon 2 and 3 segmental
amplifications of the PML-RAR allele. These primers were as follows:
IIIS, same as PS; IIIAS, 5-GCGCCGCTGCAGACTCTCGAAAAAGA-3;
IVS, 5-CCCGCTTCGGAGGAGGAGTTCCA-3; IVAS,
5-ACGAGCAGCACAGCGGCTTGGAACA-3; VS,
5-ACCCGCAAGACCAACAACAT-3; VAS,
5-CGACTGGCCATCTCCTCGTAG-3; VIS,
5-GTAGCTCACGTGCGGGCTCAGGAG-3; VIAS(1),
5-CTCTGGGCTGGCTTTCTTGGATACAG-3; VIAS(2),
5-GCGAGGGAGGGCTGGGCACTATCT-3.
The PCR segments generated from these primer sets were sequenced as
described above. PML exon 1 and 2 (partial) sequences of both alleles
were also screened by direct PCR followed by sequencing with primers
VII: VIIS, 5-CCCCTTCAGCTTCTCTTCAC-3; VIIAS,
5-TCGCACTCAAAGCACCAGAA-3. For the boundary region of
PML-RAR, primer pairs I and II, as well as VIIIS versus IAS, were
used separately in the tests of S-, V-, and L-form cases. In addition,
11 of 20 cases were also tested with primers VIIIS and VIIIAS (not
shown in Fig 1) for PML exon 7a region sequence: VIIIS,
5-CTTCCTGCCCAACAGCAACC-3; VIIIAS,
5-GCCCCAGGAGAACCCACTTT-3.
Strategy for RAR sequencing.
Bi-allelic screening of the RAR C- to F-regions was first performed
with the following two primer sets, which incorporate overlapping gene
sequences (Fig 1): IXS, 5-CCCCCTCTACCCCGCATCTACAAG-3; IXAS, 5-CGGGATCTCCATCTTCAGCGTGAT-3; XS,
5-TGATGCGGAGACGGGGCTGCTCAG-3; XAS,
5-GGGGCGGAGGGCGAGGGCTGTGTC-3. Four heterozygous base
changes detected by bi-allelic screening were further tested by nested PCR with the above-mentioned primers, after first-round PML-RAR allele-specific PCR with primer pair R: RS,
5-GCGCACCGATGGCTTCGACGAGTTC-3 and RAS (same as XAS).
| |
RESULTS |
Patient clinical characteristics.
Selected clinical features of each relapse case analyzed in this study
are summarized in Tables 1 and
2, in which the cases have been ordered on
the basis of the treatment sequence with RA and/or DA
chemotherapy. This systemization follows the two randomization points
in protocol E2491, which involved remission induction therapy with
either RA or DA and, after 2 courses of DA consolidation therapy,
re-randomization to either RA maintenance therapy for 1 year or to
simple observation (Obs).12 Cases no. 1 through 19 were
previously untreated APL patients who were enrolled on protocol E2491,
whereas case no. 20 was a non-protocol case with secondary APL that
developed 2 years after treatment of breast cancer with doxorubicin and
cyclophosphamide. The latter case as well as protocol case no. 19 (due
to the development of a toxic complication) received only RA therapy
before relapse. All other RA-treated cases received combined,
nonconcurrent DA therapy on a variety of schedules as detailed in Table
1 and below. Three of the E2491 patients failed to achieve CR on
initial protocol therapy, either because of primary drug resistance
(cases no. 12 and 15) or an intercurrent complication (case no. 16)
and, thus, are considered to be salvage cases related to the analysis of relapse samples from CRs that were achieved on alternative, cross-over therapy.
Eighteen of the E2491 patients received treatment following the four
possible protocol randomization sequences, ie, DA-Obs, DA-RA, RA-Obs,
or RA-RA. Perhaps related to a significantly higher relapse rate of
patients randomized to the DA-Obs protocol arm,12 a
disproportionate number of cases in our subset never received RA
therapy (8/19 [42%]). This group included 5 cases (no. 1 through 5)
who achieved CR on DA and were randomized to Obs after consolidation therapy; 1 case (no. 6) who was originally randomized to receive RA but
who was crossed-over to the DA arm before receiving RA; and 2 cases
(no. 7 and 8) who achieved only transient CRs and who relapsed before
completing or before beginning consolidation therapy, respectively
(Table 1). Four of the 10 combined therapy patients followed the DA-RA
treatment sequence. In cases no. 9 through 11, this was accomplished
according to protocol specification. In case no. 12, initial DA therapy
resulted in prolonged BM hypoplasia with failure to achieve CR status.
This patient was then treated off-protocol with RA and achieved a CR
after prolonged induction therapy (82 days). RA was then continuously
administered until relapse 99 days later. The RA-Obs sequence (with
intervening consolidation DA therapy) was followed in 4 cases; however,
in only 2 of these cases (no. 13 and 14) was this accomplished per
protocol. Two salvage cases (no. 15 and 16), as noted above, were also
included in this category, because they were treated with RA before
receiving any chemotherapy. These 2 cases each received an extra course of DA for cross-over CR induction in addition to the standard two
rounds of consolidation DA therapy (Table 1). Finally, 2 cases (no. 17 and 18) followed the RA-RA sequence per protocol.
In all, 12 of 20 relapse cases received oral RA therapy (45 mg/m2/d) for quite variable periods of time (19 to 415 days) before the acquisition of relapse specimens for biological and
molecular testing. In addition, 2 of these cases (no. 12 and 20) were
treated with intravenous liposomal RA (90 mg/m2 every other
day) for 72 and 15 days, respectively, after relapse from oral RA; in
these cases, biological and molecular testing were performed on samples
collected after relapse from oral RA and, additionally, after failure
of liposomal RA.
For the 19 E2491 patients detailed in Table 2, the median age was 30 years (range, 16 to 68 years); 10 cases (53%) were male and 9 cases
(47%) were female. The median pretreatment peripheral WBC count was
6,800/µL (range, 400 to 77,400/µL), and the median percentage of
the circulating blasts plus promyelocytes was 29.5% (range, 0% to
95%). In 2 salvage cases tested, the WBC count was lower than before
presentation, which may have been related to hydroxyurea administration
(case no. 15) or to partially effective ATRA therapy (case no. 16). The
percentage of cases assigned to the French-American-British (FAB) class
M3v41 was 32% (6/19 cases). Although it is not possible to
make statistical comparisons with the parent E2491/INT 0129 cohort of
221 adult PML-RAR-positive APL patients in view of the small and
incomplete number of relapse cases, it seems noteworthy that the median
values for the WBC count, the percentage of circulating blasts plus
promyelocytes, and the percentage of M3v cases were higher for the
relapse group than the corresponding median values for the overall
group.42
In vitro tests for sensitivity to RA-induced differentiation.
Among 18 pretreatment specimens tested by the NBT dye reduction test,
we found 2 cases (no. 15 and 20) with markedly reduced and 2 cases (no.
16 and 19) with moderately reduced in vitro RA sensitivity (NBT
positivity <20% at 100 nmol/L RA and <50% at 100 nmol/L RA,
respectively; Table 3). However, in the
latter 2 cases, there was no reduction in differentiation response as alternatively measured by the differentiation marker CD11b. Of 6 cases
tested after relapse from treatment with DA alone, 5 (no. 1, 2, 5, 6, and 7) had preserved and 1 (no. 8) had marginally reduced in vitro RA
sensitivity. Of 11 cases tested after relapse from regimens containing
RA in some treatment phase, 2 had preserved or slightly reduced (no. 11 and 13), 2 had moderately reduced (no. 17 and 18), and 6 (no. 9, 10, 12, 14, 16, and 20) had markedly reduced in vitro RA sensitivity
compared with pretreatment sensitivity. Neither the pretreatment nor
the relapse leukemic cells from case no. 15 were responsive to RA.
Serial testing of specimens from case no. 12 showed a progressive
decrease in RA sensitivity from pretreatment to primary DA
refractoriness to relapse from a CR achieved on oral RA to
refractoriness to intravenous liposomal RA
(Fig 2A). Complete RA dose-response curves
for the pretreatment and relapse specimens from the 3 RA-treated cases
in which mutations were found in the PML-RAR gene from the relapse
specimens (no. 9, 14, and 17; see below) are shown in Fig 2B through D.
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Table 3.
In Vitro RA-Induced Differentiation Response of
Pretreatment and Relapse APL Cells From Relapse APL Cases
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| Fig 2.
In vitro RA-induced differentiation of pretreatment and
relapse APL cells from selected patients determined by the NBT test. (A) Case no. 12; (B) case no. 9; (C) case no. 17; (D) case no. 14. ( ) pretreatment sample; ( ) relapse sample. Case no. 12 only: ( ) primary DA refractoriness; ( ) postintravenous liposomal RA.
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PML-RAR RT-PCR analysis.
The experimental strategy used for analyzing PML-RAR by RT-PCR is
shown in Fig 1 and described in Materials and Methods. As listed in
Table 4, we found the following breakdown
of PML-RAR mRNA types in our 20 relapse study cases: 6 L-form
(30%), 9 S-form (45%), and 5 V-form (25%). This is a considerable
underrepresentation of L-form cases and overrepresentation of V-form
cases, which constituted 55% and 8%, respectively, of 221 overall INT
0129 protocol cases classified by PML-RAR type.42 Ten of
14 cases (71%) analyzed expressed the reciprocal RAR1-PML fusion
gene mRNA, which approximates the percentage of protocol cases
previously reported to express RAR1-PML.43 Four of the 5 V-form PML exon 6 break/fusion site cases noted in Table 4 (no. 6, 9, 15, and 16) were previously reported.39 In case no. 9, two
additional atypical bands were noted by gel electrophoresis of RT-PCR
products from the diagnostic specimen, which were increased relative to the major V-form gel bands at relapse (Fig
3). Sequence analysis of these atypical bands showed alternative
breaksites in exon 6 at nucleotides 1576 and 1581, which coded for
truncated PML proteins, in contrast to the full-length isoform with the
break/fusion site at nucleotide 1685, which, like all other major
V-form isoforms, encoded a modified PML-RAR protein.39
In case no. 15, in which the PML exon 6 break/fusion site was also at
nucleotide 1685, a minor additional RT-PCR band was observed in 3 serial samples collected during the unsuccessful induction phase with
RA, which was shown by sequence analysis to lack all of PML exon 6 and
which encoded a truncated PML peptide (data not shown). Notably, in the
relapse specimen from this patient after a DA-induced CR, we were
unable to detect PML-RAR mRNA. No other consistent changes in the
expression pattern of PML-RAR isoforms due to alternative exon
splicing, as judged by gel electrophoretic analysis of RT-PCR product
DNAs, were observed in any other relapse specimen.
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| Fig 3.
Comparison of the gel electrophoretic pattern of RT-PCR
products from the pretreatment and relapse specimens of case no. 9. PCR
amplification used primer pair I as described in the Materials and
Methods. M1, Hae III-digested  X 174 DNA; M2, 100-bp size standard; H, HL-60 cell RNA; N, NB4 cell RNA; P, pretreatment RNA; R,
relapse RNA. PCR band 1, full-length V-form with PML breaksite at nt
1685; band 2, full-length product of comigrating atypical isoforms with
breaksites at nts 1576 and 1581; band 3, same as band 1 but lacking
exon 5 due to alternative splicing; band 4, same as band 2 but lacking
exon 5; band 5, isoform lacking exons 5 and 6 due to alternative
splicing.
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Sequence analysis of the PML-region of PML-RAR.
The primary objective of this analysis was to determine if PML-RAR
mRNA from any of the matched pretreatment-relapse APL specimens
contained base changes in PML exons 1 through 3, which are conserved in
all normal PML mRNA isoforms44 and which encode the
proline-rich, RING/B-box elements, and coiled-coil (-helical) dimerization domain of PML (see Fig 1 and Borden et al46
and de The et al47). From unambiguous, bi-directional
automated sequencing results in all 20 cases, no base changes were
observed in PML exons 1 through 3 of PML-RAR or in normal PML in
exon 1 or exon 2 sequences coding for the RING finger and first B-box (see Fig 1). Additionally, in 11 relapse cases tested, no base changes
were observed in PML exon 7a, which is conserved in all PML isoforms
and contains a casein kinase-II phosphorylation site.44
Sequence analysis of the RAR-region of PML-RAR.
Bi-allelic sequence analysis was used to investigate RAR exons 4 through 9 (C- to F-regions) as well as 16 nucleotides of the
3-UT region (Fig 1). Heterozygous base changes were detected in
4 relapse specimens, which were then further studied by allele-specific amplification and found to reside in the PML-RAR allele and not the
normal RAR allele (Fig 4). In 3 cases
(no. 9, 14, and 17), these base changes were detected in the coding
region of PML-RAR mRNA from the relapse specimen but not the
pretreatment specimen. In 1 case (no. 2), a C to T substitution was
found in the second nucleotide beyond the PML-RAR coding region stop
codon (S-form nucleotide 2478),47 ie, at the beginning of
the PML-RAR mRNA 3-UT region, in the pretreatment as well as
the relapse specimen. No base changes were observed in the RAR
B-region or PML-region adjacent to the RAR-region boundary in the
PML-RAR allele of these cases.
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| Fig 4.
Automated DNA sequence analysis of PCR products from four
APL cases with single nucleotide changes. The upper panels show bi-allelic sequencing results for the pretreatment samples, which contain normal signals as indicated by the arrows in (A), (B), and (C),
whereas (D) demonstrates a heterozygous pattern. The middle panels show
heterozygous bi-allelic signals for the corresponding relapse samples.
The lower panels show the results of nested, PML-RAR allele-specific
amplification of PCR products from the relapse cases. (A) Case no. 9, T C (MetThr). (B) Case no. 17, CT
(ArgTrp). (C) Case no. 14, CG (LeuVal).
(D) Case no. 2, CT in 3-UT region (illustrated from
antisense strand sequence analysis, ie, shown as GA; in the
upper panel the heterozygotic mutant A appears on the shoulder of the
normal G).
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Three of the PML-RAR RAR region base changes were present in the
LBD and represented point mutations resulting in amino acid codon
changes. These are described with reference to normal RAR1, because
their position is variable in PML-RAR depending on the type and
isoform of PML-RAR mRNA. In case no. 9, a T to C change was noted in
nucleotide 2745 of this shortened V-form PML-RAR mRNA (using the
sequence published by de The et al47 for L-form PML-RAR
as a reference). Relative to normal RAR1, this corresponds to
nucleotide 1340 (reference sequence, Giguere et al48) and
produces a change from methionine to threonine in codon 413 (Met413Thr). This codon is centrally located in the highly conserved
core of the activator function-2 (AF-2) activation domain (Fig
1).49 In case no. 14, a C to T change was detected in
S-form nucleotide 1955,47 which changed the codon
specificity from leucine to valine. This corresponds to RAR 1 codon
290 (Leu290Val)48 and is located more proximally in the LBD
(Fig 1). In case no. 17, a C to G transversion was detected in the
PML-RAR S-form nucleotide 2267,47 which changed the
amino acid triplet code from arginine to tryptophan. This mutational
site corresponds to RAR 1 codon 394 (Arg394Trp)48 and is
located just upstream from the conserved AF-2 activation domain (Fig
1).
| |
DISCUSSION |
A lead finding of this study was that 8 of 10 evaluable RA-treated
relapse cases, including 6 of 8 cases that relapsed after DA
consolidation therapy, had reduced APL cell sensitivity to RA-induced
differentiation in vitro (Table 3). This indicates that diminished APL
cellular RA sensitivity occurs with high frequency after relapse of
previously untreated APL cases treated with RA in combination with
intensive consolidation chemotherapy. This is similar to previously
reported findings in patients studied in second relapse after failing
both chemotherapy and RA and/or in previously untreated cases
that received less intensive postremission chemotherapy with or without
continuous maintenance RA.6,20,21 In the current study,
relapse cases with reduced RA sensitivity were seen after a variety of
RA treatment schedules, including patients who received 120 days of
RA (5/6), who received 52 days of RA (2/3), who received RA only
before DA (2/3), who received RA only after DA (3/4), who received RA
both before and after DA (2/2), who relapsed while taking RA (4/4), who
relapsed within 90 days after stopping RA (2/2), and who relapsed after
174 days after stopping RA (2/4). Clearly, with such small case
numbers it is not possible to make any conclusions about whether any of these conditions favored the development and/or maintenance of APL cellular RA resistance. However, they do suggest that there was no
tight association between the amount of RA and the development of RA
resistance and that even relatively short periods of RA treatment can
lead to durable APL cellular resistance in some cases. These results
seem remarkably different from our observation that only 1 of 6 patients treated with DA alone had reduced APL cellular RA sensitivity
(relatively minor) at relapse. These data, then, imply that RA was a
positive selective force for RA resistant relapse APL cells and,
conversely, that DA alone, as administered in protocol E2491, was not a
potent selective factor for cellular RA resistance. However, the DA
could have had a facilitative role in conjunction with RA. Our results
fundamentally disagree with early reports that relapse APL cells from
RA-treated patients retain in vitro ATRA sensitivity,16,50
which has been partly attributed to testing with a single high
concentration of RA (1 µmol/L) that could have overlooked reduced
sensitivity.14,20 Even so, in our study, 2 of 10 relapse
patients who received substantial amounts of RA had either retained or
recovered APL cell in vitro sensitivity to RA at relapse (cases no. 11 and 13), indicating that the stable loss of APL cell RA sensitivity is
not universal in RA treated patients, a finding also in accord with
previous reports.6,20,21
This study also provided some information about primary APL cell RA
resistance. When RA-naive patients have been documented to be
t(15;17)-positive and/or PML-RAR-positive, ie, to have genetically defined APL, the APL cells are almost invariably found to
be highly sensitive to RA-induced differentiation in
vitro.4,14,39 Notably, 2 of 18 of the current
PML-RAR-positive cases tested had markedly reduced sensitivity to
RA-induced differentiation in vitro at presentation (cases no. 15 and
20; Table 3). In case no. 20, a non-protocol case with secondary APL,
this seems likely related to chemotherapy with doxorubicin and
cyclophosphamide 2 years previously for breast cancer, because drug
resistance in secondary leukemias is well-recognized51 and
because diminished APL RA sensitivity has been documented after APL
relapse from extensive chemotherapy.2,21 Perhaps most
remarkably, this patient relatively promptly achieved CR (in 39 days)
that lasted for almost 1 year on RA maintenance therapy alone. This
suggests an alternative mechanism of RA-selected resistance, because
patients with APL cells resistant in vitro after relapse from RA
therapy are not frequently durably responsive to retreatment with RA in vivo.5,6,9,20,21 In case no. 15, there also may be an unusual explanation for the primary RA resistance that included failure
to respond clinically to RA induction therapy. At relapse after
achieving CR on cross-over DA therapy, the APL cells of this patient no
longer had detectable PML-RAR, as has been reported for at least 1 other documented PML-RAR-positive APL case.52 Thus, we
speculate that the lack of pretreatment APL cell RA sensitivity in this
case could have been due to an initial mixed cell or chimeric leukemia
at presentation. Consistent with this possibility, 72% of the
presentation leukemic cells were positive for CD11b antigen expression,39 which is typically expressed at very low
levels on APL cells.53 By this reckoning, the RA induction
therapy could have selected against the PML-RAR-positive
subpopulation. Against this possibility, PML-RAR mRNA remained
detectable until the time RA therapy was discontinued on day 44;
however, this is not conclusive, because PML-RAR mRNA signals
frequently remain positive by sensitive RT-PCR tests in RA-treated APL
cases through RA induction treatment and at CR.11,54 In
addition to these 2 unusual cases, 4 other cases (cases no. 9, 11, 16, and 19; Table 3) showed some apparently reduced responsiveness to 10 or
100 nmol/L RA, as measured by the NBT test, in comparison to the high degree of responsiveness of the other cases in the current patient cohort and in a larger cohort of 85 pretreatment APL
patients.39 Although it is unclear if these reductions are
significant, it seems notable that they all occurred in the RA-treated
relapse group and that 2 of these cases (cases no. 9 and 16) were
prospectively identified as possibly high-risk cases with a short
V-form of PML-RAR.39 In sum, these results suggest that
primary RA resistance or a predisposition to develop RA resistance
exists in a small subset of APL patients and that the reasons for this
are heterogeneous.
The central molecular finding of this study was point mutations in the
RAR coding region of the PML-RAR fusion gene from 3 of 12 patients after relapse from treatment regimens containing RA.
Conversely, no coding region mutations were found in APL cells from 8 patients treated with DA alone, the same chemotherapy used in a
sequential manner for induction, and/or consolidation therapy in 10 of 12 of the RA-treated patients. Although the number of cases is
too small to test statistically, the finding of mutations in 3 of 12 RA-treated versus 0 of 8 of non-RA-treated patients is consistent with
the hypothesis that the PML-RAR mutations were related to RA therapy
and to RA resistance. This hypothesis is also supported by descriptive
features of the 3 mutant cases, including the lack of PML-RAR
mutations in the pretreatment APL cells, the diminished RA sensitivity
of the relapse APL cells, and, most importantly, the nature of the
mutations in the relapse cells.
All three PML-RAR mutations were missense mutations resulting in
amino acid changes in the LBD of the RAR region. Both the Met413Thr
mutation in case no. 9 and the Arg394Trp mutation in case no. 17 constitute 2 of the 24 amino acids that have been identified by
crystallographic analysis of the RAR LBD to contribute directly to
stabilization of the RA binding pocket through the formation of
hydrogen bonds or van der Waals contacts with the bound RA
molecule.55 However, other data indicate that the Met413Thr mutation likely has only a minor effect on the binding affinity of RA
for RAR.56 Rather, the major dysfunction of this
mutation almost certainly arises because of its central location in the 7-amino acid core of the AF-2 activation domain (AD), which resides in
the 12th and final -helix of the LBD near the carboxy-terminus of
RAR.49,55 After RA binding, the 12th -helix has been
demonstrated to undergo a crucial configurational change that brings
the AF-2 AD core sequence into apposition with specific amino acid
motifs in coactivator nuclear proteins that are essential for
RAR-mediated transcriptional activation.49,55,57,58
Site-directed mutagenesis studies in which Met 413 and the adjacent
hydrophobic residue Leu 414 were deleted or replaced with the small,
neutral amino acid alanine extinguished both binding to coactivator
sequences and positive transcriptional activation
function.56-59 Although studies have not been performed
with mutations of Met 413 alone, it seems highly probable that
replacement of the hydrophobic, sulfhydryl-containing R-group of
methionine with the small polar group of threonine would be
inactivating, as was demonstrated by site-directed mutagenesis for the
nearby Met 406 residue.56 The Met413Thr mutation probably
has only a relatively minor effect on RA binding affinity because it
only comes into contact with the RA molecule after the RA-induced
configurational shift of the 12th -helix over the binding pocket,
where it serves as a lid for the presituated ligand.55 On
the other hand, the Arg394Trp mutation in case no. 17, which is
situated in the 11th -helix that also undergoes important
configurational changes on ligand binding,49,55 seems quite
likely to produce significantly altered RA binding, as was recently
reported for a nearby Leu398Pro mutation in PML-RAR in an
RA-resistant subline of NB4 cells.25 Furthermore, the amino
acid change involves a radical shift from the most hydrophilic (Arg) to
the most hydrophobic (Trp) of amino acids.60 The Leu290Val mutation in case no. 14 is located in the  -turn between -helices 5 and 6, and nearby residues in this -turn also are involved in
forming the binding pocket for RA.55 Although the amino
acid change from leucine to valine is much more conservative than the other two mutations, marked changes in protein interactions have, nevertheless, been associated with this relatively minor shift in
R-group length on hydrophobic amino acids (eg, see Heery et al58). Studies are in progress to evaluate the protein
interactions and functional properties of the three mutant PML-RAR
proteins.
In addition to the coding region mutations, a C to T change was found
in the noncoding region of PML-RAR in both the pretreatment and
relapse specimens from 1 chemotherapy treated patient whose cells
remained RA sensitive at relapse (case no. 2). This likely functionally
neutral base change raises the possibility that the development of
mutations selectively in the PML-RAR allele rather than the normal
RAR allele, both in our in vivo study and in NB4 cell line
mutants,25,28 could be related to modification of the
PML-RAR allele (or of the RAR allele involved in the translocation) in a way that increases susceptibility to mutational events.
To our knowledge, the PML-RAR mutations described here are the first
reported in this fusion gene in APL cells under naturally occurring
conditions. More generally, they appear to be the first report of
clinical mutations in an RAR sequence, aside from the integration of a
hepatitis B virus in the B-C region of RAR in a human hepatocellular
carcinoma.61 In a previous report using single-strand
conformational polymorphism (SSCP) analysis after PCR amplification
from DNA of the last 3 coding exons from the E/F region of RAR, no
RAR mutations were observed in 118 specimens from a variety of human
cell lines and fresh cells from patients with acute myeloid leukemia or
myelodysplastic syndromes.32 This sampling included 20 APL
patients, 5 of whom had acquired and 1 of whom reportedly had primary
APL cellular RA resistance, and in 7 newly diagnosed cases, the
PCR-SSCP analysis included all RAR exons encoding the B- to
F-regions. These results in no way conflict with our results that were
obtained in a highly select group of APL patients using direct
sequencing of RT-PCR products, a technique that has a lower rate of
false-negatives than PCR-SSCP analysis. Our sequencing results were
also in the great majority negative, ie, among PML-RAR-positive
relapse cases no mutations were found in the RAR region of
PML-RAR in 16 of 19 overall cases, in 8 of 11 RA-treated cases, or
in 5 of 8 RA-treated cases with diminished APL cell RA sensitivity.
Furthermore, from the bi-allelelic sequence analysis, we were able to
conclude that no mutations were present in any case in the C- through
F-regions of the normal RAR allele, which encode the DNA binding
domain, hinge/corepressor region, and the ligand binding/coactivator
domain of RAR. In addition, the PML region of PML-RAR that
specifies the proline-rich amino-terminus, the RING zinc-finger, both
B-boxes and the coiled-coil sequence, which appear to be the principal functional domains of PML and which contained two consistent mutations in an avian PML-RAR leukemia model,38 were shown to be
free of mutations. Finally, from analysis of the gel electrophoretic pattern of the RT-PCR DNA products, we found no evidence of abnormal PML-RAR splice variants that might code for competitive inhibitory proteins in any of the relapse cases that lacked PML-RAR mutations. However, in case no. 9, which harbored the Met413Thr mutation, we did
observe minor isoforms of PML-RAR due to alternative break/fusion sites in PML exon 6 that increased in the relapse APL cells (Fig 3),
and we cannot exclude the possibility that these novel isoforms, which
encode truncated PML proteins, contributed to the RA resistance in this
case. From the overall analysis, we conclude that it is improbable that
primary structural alterations of PML-RAR-encoded or normal
RAR-encoded proteins account for RA resistance in the majority of
APL relapse cases. Given the reported findings that independent
subclones of RA-resistant NB4 cells consistently form variable abnormal
protein complexes involving RA-bound PML-RAR,25-27 it
still must be considered that abnormal posttranslational modification of PML-RAR or aberrations in the other nuclear proteins that form
part of the interactive protein complex with PML-RAR could contribute to selectable APL cell RA resistance in such cases.
The apparent rarity of RAR sequence mutations in tumor cell populations
enhances the possibility that the RAR region mutations in PML-RAR |
Abstract
Introduction
Abstract