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Short Communications |
From the Department of Internal Medicine,*
Internal
Medicine and Medical Oncology, and the Department of
Biochemistry,
Biotechnology Research
Laboratories, Instituto di Ricovero e Cura a Carattere Scientifico
Policlinico S. Matteo, University of Pavia, Pavia; and the Hematology
Service,
Instituto di Ricovero e Cura a
Caraterre Scientifico Ospedale Policlinico, Milan, Italy
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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20% of
multiple myeloma patients. The translocation leads to the apparent
deregulation of two genes located on 4p16.3, the fibroblast
growth-factor receptor 3 (FGFR3), and the
putative transcription factor multiple myeloma SET
domain (MMSET), and to the generation of
IGH/MMSET hybrid transcripts. In this study, we
investigated the presence of the t(4;14) translocation in 42 AL
patients using a reverse transcriptase-polymerase chain reaction assay
for the detection of IGH/MMSET transcripts. Chimeric
transcripts were found in six patients (14%) and were consistent with
a 4p16.3 breakpoint involving intron 3 and juxtaposing
IGH regions to exon 4. In three of these cases,
hybrid transcripts juxtaposing IGH regions to exon 5
were also observed and were probably the result of an alternative
splicing skipping exon 4. Because all of the fusion transcripts (six of
six) excluded exon 3, the first translated MMSET
exon, only putative 5' truncated MMSET proteins could be
generated. In conclusion, our results demonstrate that the
t(4;14)(p16.3;q32) translocation is a recurrent genetic lesion in
primary amyloidosis.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Over the last few years, we and others have demonstrated that chromosomal translocations involving the immunoglobulin heavy chain (IGH) switch regions at 14q32 are very frequent in PC dyscrasias involving a variety of chromosome loci,2 mainly 11q13, 4p16.3, 16q23, and 6p25 where the putative target genes cyclin D1, FGFR3 and multiple myeloma SET domain (MMSET ), c-MAF and MUM1/IRF4 are respectively located.3-8 The t(4;14)(p16.3;q32) translocation is of particular interest because it seems to be specifically associated with multiple myeloma (MM) (20% of cases),9 and leads to the apparent deregulation of two potential proto-oncogenes, FGFR-3 (fibroblast growth factor receptor 3)4,5 and MMSET.6 It also leads to the formation of fusion IGH/MMSET hybrid transcripts, which are specific molecular markers that can be detected by reverse transcriptase-polymerase chain reaction (RT-PCR).6,9
In this study, we investigated the presence of the t(4;14) translocation in primary AL by using a recently described sensitive RT-PCR assay9 to look for related IGH/MMSET transcripts in bone marrow taken from 42 patients.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The patient population consisted of 42 randomly chosen patients
with primary AL who underwent bone marrow aspiration at the
coordinating center of the Italian Amyloid Program (Pavia, Italy).
Amyloid was identified by means of Congo-red staining on tissue
biopsies and/or abdominal fat aspirates taken after the patients had
given their informed consent. Marrow PC clonality was assessed by means
of double-staining immunofluorescence on Ficoll-separated mononuclear
cells using fluorochrome-conjugated anti-light-chain isotype antisera
(DAKO, Glostrup, Denmark); clonality is indicated by a 
/
isotype
ratio <1.1 ( PC clone) or >2.6 ( PC clone).10
The
patients showed a monoclonal component at serum or urine
immunofixation using anti-isotype-specific rabbit-antisera on
high-resolution agarose gel electrophoresis (DAKO).10
Any
association with clinically overt MM (percentage of PC >15% and renal
failure or hypercalcemia or osteolytic bone lesions) was excluded by
clinical and laboratory findings. There was no family history
suggestive of hereditary AL in any patient.
Bone marrow from 11 normal donors, 9 patients with secondary thrombocytosis, 11 patients with primary thrombocythemia, and 3 patients with polycythemia vera were investigated as negative controls for the presence of the IGH/MMSET transcripts.
RT-PCR Analysis of IGH/MMSET Hybrid Transcripts and MMSET Gene Expression
Total RNA from the Ficoll-separated bone-marrow mononuclear cells
of the AL patients, PC lines (KMS-11, NCI-H929, and OPM-2, used as
positive controls for the three known translocation breakpoints), and
34 negative controls was extracted using Trizol (Life Technologies,
Inc., Grand Island, NY). RT-PCR analysis was performed as
described previously.9
Briefly, 1 µg of total RNA was
transcribed using Superscript RT (Gibco-BRL) and random hexamers
(Pharmacia Biotech, Uppsala, Sweden). The first PCR round was performed
using a portion of the first-strand cDNA as template, JH
(5'-CCCTGGTCACCGTCTCCTCA-3') or Iµ1 (5-AGCCCTTGTTAATGGACTTG-3') as 5'
primers, and the 3' primer mmset exon 6 reward
(ms6r) (5'-CCTCAATTTCCCTGAAATTGGTT-3') (see Figure 1
for primer positions). For nested-PCR,
1 µl of the first PCR round was re-amplified using Iµ2
(5-CTTTGCAAGGCTCGCAGTGAC-3') and ms5r
(5-AAGAACTGTACGTGATACT-3') as internal primers (Figure 1)
. The PCR
products were electrophoresed on a 1.8% agarose gel in Tris
borate-ethylenediaminetetraacetic acid and visualized by staining with
ethidium bromide.
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DNA Sequencing
Direct DNA sequencing was performed on PCR-amplified fragments using the appropriate primers described above. The DNA fragments were purified by agarose gel extraction (QIA quick kit; Qiagen, Valencia, CA) and sequenced in both directions using an automated DNA sequencer (Applied Biosystems, Foster City, CA) and the Taq Dye Deoxy Terminator cycle sequencing kit (Perkin-Elmer, Norwalk, CT).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Table 1
summarizes the clinical
findings and laboratory values of the 42 AL patients included in our
study. Median PC infiltration was 7%. Expansions of monoclonal PCs
were detected by means of bone marrow light-chain isotype ratio
analysis in 37 cases (88%) and a monoclonal component was identified
at serum and urine immunofixation on high-resolution agarose gel
electrophoresis in 93% of the patients. These results are compatible
with the sensitivities of the methods used,10
and a
combination of the two analyses10
confirmed a monoclonal
disorder in all cases. Twenty-four of the 42 patients were alive at the
time of our study, the others having died from AL-related causes.
Median survival was 32 months.
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The t(4;14)(p16.3;q32) chromosomal translocation leads to the
juxtaposition of the IGH locus to the MMSET gene
in the same transcriptional orientation on both der(4) and der(14)
chromosomes.6
4p16 breakpoints occur within the 5' introns
of the MMSET gene or upstream of its coding sequence
(probably in its regulatory regions); three different types of
breakpoints have so far been characterized in MMs (Figure 1)
.6,9
The translocation leads to hybrid
JH-MMSET and Iµ-MMSET transcripts
from der(4) chromosome, probably initiating from the VDJ or Iµ
(intron µ) promoters (Figure 1)
. Hybrid MMSET-IGH
transcripts from the reciprocal der(14) have been less frequently
identified.6
The presence of IGH-MMSET fusion transcripts was
investigated in the panel of 42 patients using the previously described
RT-PCR assay. No hybrid transcripts were detected in the first PCR
round using both sets of IGH-MMSET primers
(JH-msr6 or Iµ-msr6), a finding that is
consistent with the scarce PC infiltration typical of primary AL.
Nested-PCR (sensitivity,
10-5) was then performed
using the Iµ2 and ms5r internal primers on the fragments
obtained using the Iµ1 and ms6r primers (Figure 1)
. Figure 2
shows the results from the six positive
cases for IGH-MMSET rearrangements. A breakpoint-type 2
transcript was detected in three patients (Figure 2
, lanes A to C),
whereas transcripts corresponding to b-type 2 and b-type 3 were
observed in the other three cases (Figure 2
, lanes D to F). Finally, no
hybrid IGH/MMSET transcripts were detected by single-round
and nested RT-PCR in RNAs from the bone marrow of 34 negative controls.
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Clinicopathological Features of the Patients with a t(4;14) Translocation
Table 2
shows the
clinicopathological features of the patients harboring the t(4;14)
translocation. Two patients (Table 2
, PG and AR) had long survival
times (36 and 63 months) and no subsequent MM was observed. In two
cases (Table 2
, MG and AR), the PC clone was very small and the PC
light chain isotype ratio was normal, a situation that is observed in
15% of AL patients.10,11
Therapy consisted of
melphalan and prednisone in all but nine cases treated with high-dose
dexamethasone.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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20% of MMs and
leads to the apparent deregulation of the FGFR3 and
MMSET genes located on 4p16.3.4-6,9
Interestingly, it also leads to the formation of hybrid
IGH/MMSET transcripts initiating from (V)DJ and Iµ
IGH promoters relocated on chromosome
der(4);6,9
reciprocal MMSET/IGH transcripts
from der(14) have also been found but significantly less frequently. It
has been predicted that most of the chimeric IGH/MMSET
transcripts do not encode putative fusion proteins,6
but
full-length MMSET proteins if the breakpoint is upstream of exon 3
(b-type 1), or 5' truncated MMSET proteins respectively lacking the
terminal 238 or 323 amino acids if the breakpoints are upstream of exon
4 (b-type 2) or 5 (b-type 3) (Figure 1)
; in-frame AUG codons are
present in exons 3, 4, and 6.6
The recent availability of a specific and reliable (fully fluorescence
in situ hybridization-concordant) RT-PCR assay (single-round
or nested PCR) for detecting IGH/MMSET hybrid transcripts
from all of the of 4p16.3 breakpoints so far identified in
MM9
prompted us to search for t(4;14) in a panel of
samples from 42 patients with primary AL. Given the scarce PC
infiltration in AL, this assay should be considered a method of choice
for detecting the possible presence of t(4;14). The patient series was
fairly representative of a typical AL population in terms of PC
infiltration, the distribution of organ involvement and
survival.14
IGH-MMSET hybrid transcripts were
detected only by means of nested RT-PCR in 6 of the 42 patients (14%):
the fusion transcripts involved MMSET exon 4 (b-type 2) in
three cases and exon 4 and exon 5 (b-type 2 and 3) in the remaining
three cases (Table 2
and Figure 2
). None of the fusion transcripts
included exon 3 (b-type 1). In a previous series of myeloma patients
investigated using the same RT-PCR assay,9
6 of 11 hybrid
transcripts (54%) were type 1, whereas the only positive MGUS case had
a b-type 2 transcript like our amyloid patients. Thus, if the various
full-length and 5' truncated MMSET proteins were translated from
chimeric transcripts, these amyloid cell clones would generate 5'
truncated MMSET proteins from translocated alleles.
Concerning the three patients showing both the b-type 2 and 3
transcripts (Table 2
and Figure 2
, lanes D to F), these data were
confirmed by direct sequencing (data not shown) and represent a novel
finding because none of the 11 previously described MM patients with a
t(4;14) translocation showed this pattern.9
One possible
explanation is that these transcripts represent different mRNA spliced
forms of the MMMSET gene from a b-type 2 translocation.
Although IGH/MMSET transcripts were only detected by means of nested RT-PCR (which is consistent with the low level of PC infiltration in our patients), this finding raised the question as to whether this highly sensitive method (10-5) could detect hybrid transcripts from t(4;14) that may occur in the hematopoietic cells of normal individuals, as has been demonstrated for the t(14;18) chromosomal translocation.15 Although we did not address this question specifically, our analysis showing no IGH/MMSET transcripts in bone marrows from 34 negative controls supports the validity of the nested RT-PCR approach in AL.
The analysis of various clinicopathological features in the six positive patients did not reveal any apparent correlation with the presence of fusion transcripts. However, because the number of positive cases was small, any clinicobiological relevance of this alteration remains to be elucidated in larger studies. This could be relevant because, although survival in AL is related to organ dysfunction,14 it is also influenced by clone dimensions11 and therapeutic response; biological variables are therefore needed to identify categories of patients whose worse prognosis warrant more aggressive therapies.
In conclusion, this study shows that the t(4;14) translocation is a recurrent lesion in AL patients (6 of 42, 14%). Its incidence seems to be higher than in MGUS (1 of 16 positive, 7%) and slightly lower than in MM (11 of 53 positive, 20%).9 Taken together, these findings further support the concept that IGH translocations are common and relevant genetic events in PC dyscrasias.
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Footnotes |
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Supported by grants from Associazione Italiana per lu Ricerca sul Cancro (to A. N. and G. M.), European Biomed 2 (program no. BMH4-CT 98-3689 to G. M.), Progetto di Ateneo, Ministero della Università e della Ricerca Scientifica e Tecnologica 1999 (nos. 9906038391-010 and 9906038391-007 to A. N. and G. M., respectively), Fondazione Ferrata-Storti, and Instituto di Ricovero e Cura a Carattere Scientifico Policlinico S. Matteo.
Accepted for publication February 8, 2001.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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) used for
the first (JH or Iµ1 plus
ms6r) and second
(Iµ2 plus
ms5r) RT-PCR rounds are shown below
the map. The PCR fragment length localizes the 4p16.3 breakpoint. The
figure is not to scale.