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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 94 No. 5 (September 1), 1999:
pp. 1755-1760
Mutation of the p53 Gene Is Not a Typical Feature of Hodgkin and
Reed-Sternberg Cells in Hodgkin's Disease
By
Manuel Montesinos-Rongen,
Axel Roers,
Ralf Küppers,
Klaus Rajewsky, and
Martin-Leo Hansmann
From the Institute for Genetics, University of Cologne, Cologne; and
the Institute for Pathology, University of Frankfurt, Frankfurt,
Germany.
 |
ABSTRACT |
Point mutations of the p53 tumor suppressor gene are a frequent
finding in human carcinomas and are thought to be an important oncogenic event. In non-Hodgkin lymphomas, p53 mutations occur in a
minor fraction of cases. However, conclusive data are still lacking for
Hodgkin's disease (HD) where the analysis meets technical problems.
The neoplastic tumor cell clone in HD is represented by the large
Hodgkin and Reed-Sternberg (HRS) cells, which account for only a
minority of all cells in the tumor tissue (often <1%). To identify
putative HRS cell-specific mutations, single HRS cells were
micromanipulated from frozen tissue sections of HD biopsy specimens.
Exons 4 to 8 of the p53 gene (in which more than 90% of p53 mutations
associated with human neoplasms occur) were amplified from these single
cells and sequenced. Mutations of p53 were not found in HRS cells of
any of 8 cases of HD analyzed. We conclude that mutation of the p53
gene is only rarely, if at all, involved in the pathogenesis of HD.
© 1999 by The American Society of Hematology.
| |
INTRODUCTION |
HODGKIN AND REED/STERNBERG (HRS) cells
represent the malignant tumor cells of Hodgkin's disease (HD). In
contrast to most other neoplasms, the tumor clone in HD accounts for
only a minor fraction of all cells in the tumor tissue.1 It
has recently been shown that the HRS cell population is derived from mature B cells.2 The presence and pattern of somatic
mutations in rearranged immunoglobulin variable region genes amplified
from HRS cells suggested that the HRS cell precursor is a germinal center (GC) B cell which has lost the capacity to express antigen receptor. GC B cells with "crippled" antigen receptor die by
apoptosis under normal circumstances. The HRS cell precursor might
initiate the apoptotic pathway, but survive because of an unknown
transforming event.2,3
Loss of function of the p53 protein seems to be important in the
oncogenesis of human cancer because inactivating point mutations of the
p53 gene are found in a high proportion of colorectal, lung, and breast
cancers.4 In carcinoma cells, mutation of one allele is
usually accompanied by deletion of the second allele.5,6 Mutations of the p53 tumor suppressor gene have also been investigated in lymphomas and were found in a minority of non-Hodgkin lymphomas (NHL) on primary disease manifestation, but with higher frequency in
relapsed cases.7-9
Whether alteration of the p53 gene plays a role in malignant
transformation of HRS cells in HD is a long debated question. The
search for mutations in the DNA of HRS cells is hampered by the
scarcity of these cells in the tumor tissue (often <1% of all
cells). Mutations have been detected in a minor fraction of HD cases by
the single-strand conformation polymorphism (SSCP) technique in DNA
extracted from whole-tissue specimens.10-16 However, it was
not possible to unequivocally show that the mutated sequences indeed
originated from the HRS cell population. By amplification of p53 cDNA
from single HRS cells isolated from cell suspensions of fresh tumor
tissue in 3 cases of HD, Trümper et al showed that (part of) the
HRS cell clone of 3 patients carried a mutated p53
allele.17,18
To investigate whether p53 mutations frequently occur in HD, we
micromanipulated single HRS cells from frozen tissue sections of 8 patients suffering from classical HD. Exons 4 to 8 of the p53 gene,
where more than 90% of mutations of p53 are found,4,7 were
amplified from the genomic DNA of these single cells in a multiplex
polymerase chain reaction (PCR) and sequenced.
| |
MATERIALS AND METHODS |
Tissue samples and cell line CO.
Patient data are summarized in Table 1. In
all cases of primary HD, enlarged cervical lymph nodes were analyzed
except for case 8, which was an axillary lymph node. These cases were
selected to represent the typical histology of nodular sclerosing (NS) or mixed cellularity (MC) HD. The material of the 2 relapsed cases, 6 and 7, was a paraaortal and an inguinal lymph node biopsy,
respectively. In all 8 cases, the HRS cells were positive for CD30 and
except for 1 case (relapsed case 6), the HRS cells also expressed CD15. (No material for CD15 staining was available for case 4 [Table 1].)
Cell line CO10,19 was obtained from V. Diehl (University of
Cologne, Cologne, Germany).
Sorting of peripheral blood cells.
Peripheral blood mononuclear cells (PBMC) were isolated from blood of a
healthy Caucasian donor by centrifugation over Ficoll Paque R
(Pharmacia, Freiburg, Germany) and stained with anti-  -TCR-PE (Immunotech, Hamburg, Germany) and anti-CD3-FITC (Becton Dickinson, Heidelberg, Germany). Single -TCR+/CD3+
cells were deflected into PCR tubes using a FACS 440 (Becton Dickinson)
as described.20
Immunostaining of frozen tissue sections and cytospins and
micromanipulation.
Eight-micrometer-thick frozen sections of tumor tissue
were mounted onto glass slides, air-dried, and fixed in acetone for 10 minutes. PBMC were mixed with CO cells, centrifuged onto glass slides,
air-dried, and fixed in acetone for 10 minutes. Sections and cytospins
were stained with antibodies against p53 (CM1; NovoCastra, Newcastle-Upon-Tyne, UK), CD15 (LeuM1; Becton Dickinson), CD20 (L26;
Dako, Glostrup, Denmark) or CD30 (BerH2; Dako) as described previously.20 Sections were overlaid with Tris-buffered
saline containing 5 mg/mL collagenase H (Boehringer Mannheim, Mannheim, Germany). Single cells were mobilized from the sections with the help
of hydraulic micromanipulators (Narishige, Japan) as
described.21
Amplification of exons 4 to 8 of the p53 gene from single cells.
Single cells in 20 µL Expand HF PCR buffer (Boehringer Mannheim) were
incubated with 0.25 mg/mL proteinase K (GIBCO Life Technologies, Eggenstein, Germany) for 2 hours at 50°C. The enzyme was inactivated by heating to 95°C for 10 minutes. The first round of amplification was performed in the same reaction tube in a 50 µL volume containing 1X Expand HF buffer; 2 mmol/L MgCl2, 125 µmol/L each of
dATP, dCTP, dTTP, and dGTP; 16.6 nmol/L each first round primer (4UA, 4D, 56UA, 56D, 78UA, and 78D; Table 2); and
2.5 U of Expand HF polymerase mix (Boehringer Mannheim). Thermal
cycling was performed in a Biometra Unoblock (Biometra,
Göttingen, Germany). An initial cycle of 95°C for
2 minutes, a pause at 80°C during which the enzyme was added, 61°C
for 30 seconds, and 72°C for 50 seconds was followed by 35 cycles of
95°C for 60 seconds, 61°C for 30 seconds, and 72°C for 50 seconds, followed by 72°C for 5 minutes. For the second round of
amplification, 1 µL of the first-round reaction mixture was added to
3 separate reaction mixes each containing 1 primer pair (4UB and 4D,
5/6UB and 5/6D, 7/8UB and 7/8D; Table 2). Second-round reactions were
performed in a volume of 50 µL with 20 mmol/L Tris-HCl, pH 8.4, 50 mmol/L KCl, 2 mmol/L MgCl2, 125 µmol/L each of dATP,
dCTP, dTTP, and dGTP, 125 nmol/L each primer and 1.25 U Taq DNA
polymerase (GIBCO) in 96-well plates (Costar, Bodenheim, Germany) using
a Biometra Uno-Thermoblock. All components were added on
ice and the 96-well plate was placed into the thermocycler after the
block had reached denaturing temperature (95°C). The cycle program
consisted of 1 cycle of 95°C for 3 minutes, 61°C for 30 seconds,
and 72°C for 90 seconds, followed by 44 cycles of 95°C for 60 seconds, 61°C for 30 seconds, and 72°C for 60 seconds, followed by
a 5-minute incubation at 72°C.
Sequence analysis.
PCR products were directly sequenced from both sides using the
second-round primers. Cycle sequencing was performed using the Ready
Reaction Dye Deoxy Terminator cycle sequencing kit (Perkin Elmer,
Foster City, CA) and an ABI 377 automatic sequencer. DNASIS software
(Pharmacia) and the GenBank data library (release 93.0) were used to
analyze the sequences.
| |
RESULTS |
Sections of paraffin-embedded tissue specimens were immunostained for
p53 protein in 6 of the 8 cases investigated. In all 6 cases, at least
80% of the HRS cells were positive for p53 protein (Table 1).
Because more than 90% of all point mutations found in the p53 gene of
malignant cells are located within the downstream part of exon 4 and in
exons 5 to 8,4,7 sequence analysis was focused onto these
exons. Amplification of exon sequences from single cells was performed
by seminested PCR. The primers hybridized in the intronic sequences
flanking the exons. Three segments of the p53 gene were amplified with
different primer pairs, the first (E4) containing the downstream part
of exon 4, the second (E5/6) containing exons 5 and 6, and the third
(E7/8) containing exons 7 and 8. In the first round of amplification,
all 6 primers were used together in the same tube, while second round
amplifications were performed in separate reactions. As a positive
control for efficient amplification from single genomes, single cells
(peripheral blood T cells from a healthy donor) were sorted into PCR
tubes by flow cytometry and analyzed by PCR. All 3 segments of the p53 gene could be amplified from 81 of 87 tubes (90%). The remaining 6 tubes were negative for all 3 products, which is most likely because of
failure of the single-cell deflection.
Potential alterations of the p53 gene in HD may occur as point
mutations on 1 allele with concomittant loss of the second allele. This
is typically found in carcinomas. Alternatively, the 2 alleles may be
affected by 2 different point mutations. In the latter situation,
mutations are more difficult to detect by amplification and sequencing
because mixed sequences result if both alleles are amplified from a
given cell. To validate that the method used in the present study can
detect a point mutation in micromanipulated single cells, which also
harbor an unmutated allele of the p53 gene, a control experiment was
performed using cell line CO. CO cells harbor 2 mutated
alleles10 with 1 point mutation located in exon 5 and a
second in exon 8 (codons 175 and 282, positions 13203 and 14513 of the
published wild-type sequence of the p53 gene,22 accession
no. X54156). Cells of the CO line are large and express CD30 but not
CD20. CO cells were mixed with PBMC from a healthy donor in a ratio of
1:100 and the mixture was centrifuged onto glass slides such that the CO cells were tightly surrounded by PBMC. Cytospins were stained for
CD30 or CD20. Large CD30+ cells were micromanipulated from
CD30-stained cytospins and analyzed by single-cell PCR. Small
CD20+ cells manipulated from CD20-stained cytospins served
as controls. Exon pairs 5/6 and 7/8 could be amplified from 7 of 10 CD30+ cells. For the majority of the CD30+
cells, the sequencing chromatograms showed a double band at positions 13203 and 14513, indicating coamplification of both the mutated and the
unmutated sequence (Table 3). The double
band was detectable unambigously in forward and reverse sequencing
reactions. In rare instances, only the mutated sequence was obtained
(Table 3). The CD20+ control cells yielded unmutated
sequences only. These results show that the combination of
micromanipulation and single-cell PCR reliably detects point mutations
in single cells even if the cells are heterozygous for this mutation.
Tissue samples from 8 cases of HD were analyzed, 6 of which were biopsy
specimens taken at primary manifestation of the disease. Two biopsy
specimens were taken at first relapse (Table 1). HRS cells were
identified in frozen sections of tumor tissue as large, multinucleated
CD30+ cells. Ten to 12 HRS cells were micromanipulated for
each case and subjected to single-cell amplification. To be able to
identify HRS cell-specific mutations, non-HRS cells (CD20+
B cells) were micromanipulated from adjacent sections and analyzed in
parallel as controls. Aliquots of the buffer covering the section during the micromanipulation procedure served as negative controls in
the PCR. These controls were always negative except for 1 aliquot in
case 4 from which all 3 p53 gene segments were amplified. This was
probably due to cellular contamination during the micromanipulation procedure because 14 additional buffer aliquots from the same case were
negative. Each of the 3 p53 gene segments (E4, E5/6, E7/8) was
amplified from at least 4 different HRS cells (with the exception of
segment E7/8 in case 8; Table 4). In most
cases, each gene segment was amplified from 6 to 8 different HRS cells. PCR products were directly sequenced from both sides.
No sequence differences were detected between HRS and B cells in the
downstream part of exon 4, over the complete length of the coding
regions of exons 5 to 8 and the exon-intron boundaries in any of the 6 primary nor the 2 relapsed cases analyzed. All exonic sequences
amplified from the HRS and B cells were identical to the published
sequence of the p53 gene.22 The chromatograms were also
analyzed for double bands indicating heterozygosity at a particular
position; however, no mixed sequences could be identified.
In 4 of the 8 cases (case 1, 2, 3, and 8), sections of the same tissue
samples had previously been used for an analysis of the HRS cell
population for the presence of immunoglobin (Ig) gene rearrangements
(case 2, 5, 7, and 4 in Kanzler et al,2 respectively) by
single-cell PCR. In all these cases, the CD30+ HRS cells
identified using the same criteria as applied in the present study and
micromanipulated under the same conditions, were shown to harbor clonal
Ig gene rearrangements.2 In the present study, the
VH3 gene rearrangement of the HRS cell clone of case 8 was
coamplified along with the p53 exons to provide direct evidence that
the p53 PCR products originated from the clonal HRS cell population of
this case. This was done by addition of a VH3-specific and
a JH-specific primer (primers VH3 and
3'JH6)2 to the reaction mix in the first
round of amplification. The clonal VH3 rearrangement was
obtained from 6 of 16 HRS cells (Table 5). Five of 8 HRS cells from which unmutated p53 PCR products were obtained
were also positive for the clonal VH3 gene rearrangement. One cell was excluded from the sequence analysis because a clonally unrelated VH3 gene rearrangement was coamplified with the
p53 exons. This cell likely represents cellular contamination. These results show that the majority of the p53 PCR products analyzed indeed
originate from the clonal population of HRS cells.
| |
DISCUSSION |
GC B cells are preprogrammed to die by apoptosis unless rescued by a
signal through the antigen receptor.23 HRS cells seem to be
derived from GC B cells that have "crippled" their antigen receptor genes by somatic mutations.2,3 Therefore, an
intriguing question is how these cells are rescued from undergoing
apoptosis. Epstein-Barr virus can immortalize B cells and in about half
of the cases of HD the HRS cells are infected with this
virus.24 So far, however, there is no direct proof that
apoptosis of the HRS cell precursor might be blocked by infection with
Epstein-Barr virus.
A molecular feature common to many, if not all, HD cases is an abnormal
accumulation of NF B in the nuclei of HRS cells.25,26 This might be caused by defective IB or blocked expression of this
inhibitor of NFB.27 Constitutive nuclear expression of NFB has been shown to render HD-derived cell lines resistant to
apoptosis-inducing stimuli and may, therefore, play a role in the
rescue of crippled GC B cells from programmed cell death.26
The p53 gene is induced after DNA damage. Apart from arresting cells in
the G1 phase of the cell cycle, p53 protein can also induce
apoptosis.28,29 To find out whether inactivation of this
protein might be one of the oncogenic events that renders HRS cell
precursors resistant to apoptosis, we analyzed p53 genomic sequences
from single HRS cells of 8 cases of HD by a combination of
micromanipulation and single-cell PCR. A validation experiment proved
the reliability of this approach. CO cells19 were mixed with PBMC from a healthy donor and the mixture was centrifuged onto
glass slides. After micromanipulation of single CO cells and B cells
from immunostained cytospins, the p53 mutations known to be present in
the genome of CO cells10 were detected in all products
amplified from the single CO cells but not from the control B cells. In
contrast, for all 8 cases of HD analyzed, only unmutated p53 sequences
were amplified from single micromanipulated HRS cells.
Several earlier studies used the SSCP technique to search for mutated
p53 genomic sequences in DNA extracted from HD tumor tissue.11-16 Mutated p53 sequences were detected in a minor
fraction of cases. Chen et al,14 followed a more refined
approach. They microdissected and analyzed by PCR and SSCP areas in
sections of tumor tissue that contained more than 10% HRS cells.
Mutated p53 sequences were detected in 6 of 23 cases. However, the SSCP technique does not permit to attribute mutated sequences to the HRS
cell population.
In 2 studies, an attempt was made to selectively analyze HRS cells for
p53 mutations.10,17,18 Gupta et al10 enriched HRS cells by sorting large (presumably polyploid) cell nuclei from cell
suspensions prepared from paraffin-embedded biopsy specimens of 2 patients. p53 genomic sequences were then amplified in a 2-rounded PCR.
Sequence heterogeneities were detected by SSCP analysis and were
assumed to be due to mutations in the HRS cell population. However,
direct proof for this was missing, and it was a peculiar and
unexplained finding that only some of the sequences obtained from the
cloned aberrantly migrating PCR products indeed harbored a mutation. In
addition to the material of these 2 cases of primary HD, Gupta et al
also analyzed the p53 genes of 6 HD-derived cell lines. p53 mutations
were found only in cell line CO. However, in light of our earlier
finding that in most, if not all cases of HD, HRS cells harbor clonal
Ig gene rearrangements,2 it is questionable whether cell
line CO that carries Ig loci in germline configuration30 is
derived from HRS cells.
Trümper et al17,18 analyzed 3 cases of HD for HRS
cell-specific p53 mutations by micromanipulating single CD3-, CD20-, CD14-negative, or CD30-positive cells with HRS cell morphology from
cell suspensions of fresh tumor tissues. p53 cDNA was amplified and
cloned PCR products were sequenced. In each of the 3 cases, some of the
cells harbored identical point mutations, whereas p53 sequences from
other cells of those cases were unmutated. These data were interpreted
as indication for clonal heterogeneity of HRS cells.
Our finding of only unmutated sequences in HRS cells from 8 cases is in
contrast to the results of Trümper et al who found mutated p53
sequences in the majority of HRS cells in all 3 cases analyzed. We
cannot explain this discrepancy. The combination of micromanipulation
and single-cell PCR used in the present study has previously proven to
reliably yield HRS cell-specific sequence information. In 24 of 25 cases of HD, clonal Ig gene rearrangements were obtained from HRS cells
that had been identified by the same pathologist using the same
histological criteria and that had been micromanipulated and analyzed
exactly as in the present study (including cases 1, 2, 3, and 8 of the
present study).2,31-36 In the only case that was negative
for Ig gene rearrangements, this result was later found to be because
of insufficient DNA quality. For case 8 of the present study, we have
provided direct evidence that the unmutated p53 sequences were indeed
amplified from HRS cells and not from contaminating non-HRS cells by
coamplification of the HRS clone-specific IgH gene rearrangement of
this case. Our data do not rule out the possibility that in a minor
fraction of HD cases, some or all HRS cells carry mutated p53 alleles. A mutation of the p53 gene present in only a minor HRS cell subclone may escape detection by analysis of a limited number of single micromanipulated HRS cells, but could potentially be detected in
SSCP-based analyses. However, if only part of the HRS cell clone
carries mutated p53 alleles, these mutations must have occurred late in
disease progression and are, therefore, not characteristic events that
contribute to malignant transformation of HRS cell precursors in HD.
In 6 of the 8 cases analyzed, at least 80% of the HRS cells were
positive for p53 by immunohistochemistry. Thus, most p53 sequences
amplified from HRS cells originate from this large fraction of
p53-expressing HRS cells. It has been shown that certain point mutations in exons 4 to 8 of the p53 gene can increase the half-life of
p53 protein.37 In HD, increased half-life of p53 protein was discussed as a possible reason for the increased expression levels
of p53 found in HRS cells of a high proportion of cases.38 Our data suggest that mechanisms other than mutation of the p53 gene in
exons 4 to 8 must be responsible for upregulation of p53 protein in HRS cells.
| |
ACKNOWLEDGMENT |
We thank Julia Jesdinsky and Arianne Faßbender for expert technical assistance.
| |
FOOTNOTES |
Submitted September 7, 1998; accepted April 28, 1999.
M.M-R. and A.R. contributed equally to this work.
Supported by the Deutsche Forschungsgemeinschaft through SFB502.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Axel Roers, MD, Institute for
Genetics, University of Cologne, Weyertal 121, 50931 Cologne, Germany.
| |
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