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.

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.5,6Mutations 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).

Table 1.

Patient Data

Case HD Manifestation HD Subtype Age Sex HRS Cells
CD30CD15 p53
1  Primary  NS  51  Female  +  +  
2  Primary  NS  27  Female  +  +  
3  Primary  MC  36  Male  +  +  ND  
Primary  NS  17  Female  +  ND  +  
Primary  MC  69  Male  +  +  +  
6  First relapse  ?* 40  Male  +  −  +  
7  First relapse  MC  17  Male  +  +  ND  
8  Primary NS  18  Female  +  +  
Case HD Manifestation HD Subtype Age Sex HRS Cells
CD30CD15 p53
1  Primary  NS  51  Female  +  +  
2  Primary  NS  27  Female  +  +  
3  Primary  MC  36  Male  +  +  ND  
Primary  NS  17  Female  +  ND  +  
Primary  MC  69  Male  +  +  +  
6  First relapse  ?* 40  Male  +  −  +  
7  First relapse  MC  17  Male  +  +  ND  
8  Primary NS  18  Female  +  +  

Abbreviations: HD, Hodgkin’s disease; ND, not done; NS, nodular sclerosing; MC, mixed cellularity.

*

Classical HD, subtype of primary manifestation unknown.

“+” indicates that at least 80% of HRS cells are positive for p53 protein.

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–αβ-TCR-PE (Immunotech, Hamburg, Germany) and anti–CD3-FITC (Becton Dickinson, Heidelberg, Germany). Single αβ-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.

Table 2.

Primers for Amplification of Exons 4 to 8 of the p53 Gene From Single Cells

4UA  5′-ACT-GAA-GAC-CCA-GGT-CCA-GAT-GAA-3′  
4UB 5′-TTC-TGT-CCC-TTC-CCA-GAA-AAC-CTA-3′  
4D 5′-AGG-GTG-AAG-AGG-AAT-CCC-AAA-GTT-3′  
5/6UA 5′-TGT-TCA-CTT-GTG-CCC-TGA-CTT-TCA-3′  
5/6UB 5′-CTC-TGT-CTC-CTT-CCT-CTT-CCT-ACA-3′  
5/6D 5′-AGG-GCC-ACT-GAC-AAC-CAC-CCT-TA-3′  
7/8UA 5′-CAA-GGC-GCA-CTG-GCC-TCA-TCT-T-3′  
7/8UB 5′-CCT-CAT-CTT-GGG-CCT-GTG-TTA-TCT-3′  
7/8D 5′-ACC-GCT-TCT-TGT-CCT-GCT-TGC-TTA-3′ 
4UA  5′-ACT-GAA-GAC-CCA-GGT-CCA-GAT-GAA-3′  
4UB 5′-TTC-TGT-CCC-TTC-CCA-GAA-AAC-CTA-3′  
4D 5′-AGG-GTG-AAG-AGG-AAT-CCC-AAA-GTT-3′  
5/6UA 5′-TGT-TCA-CTT-GTG-CCC-TGA-CTT-TCA-3′  
5/6UB 5′-CTC-TGT-CTC-CTT-CCT-CTT-CCT-ACA-3′  
5/6D 5′-AGG-GCC-ACT-GAC-AAC-CAC-CCT-TA-3′  
7/8UA 5′-CAA-GGC-GCA-CTG-GCC-TCA-TCT-T-3′  
7/8UB 5′-CCT-CAT-CTT-GGG-CCT-GTG-TTA-TCT-3′  
7/8D 5′-ACC-GCT-TCT-TGT-CCT-GCT-TGC-TTA-3′ 
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.

Table 3.

Validation Experiment: Detection of p53 Point Mutations in Single Micromanipulated CD30+ CO Cells

Cell Exon 5 (codon 175) Position 13203 Exon 8 (codon 282) Position 14513
3-150CD30+ 
  1  A/G  T/C  
  2  A/G T/C  
  4  A/G  T/C  
  7  A/G  T/C  
  8 A  T/C  
  9  A/G  T/C  
 10  A  
3-151CD20+ 
 36  G  C  
 38  G  
 39 G  C  
 40  G  C  
 41  G  C  
Wild-type p533-152 G  C  
Cell line CO  A  
Cell Exon 5 (codon 175) Position 13203 Exon 8 (codon 282) Position 14513
3-150CD30+ 
  1  A/G  T/C  
  2  A/G T/C  
  4  A/G  T/C  
  7  A/G  T/C  
  8 A  T/C  
  9  A/G  T/C  
 10  A  
3-151CD20+ 
 36  G  C  
 38  G  
 39 G  C  
 40  G  C  
 41  G  C  
Wild-type p533-152 G  C  
Cell line CO  A  
F3-150

CO cells.

F3-151

B cells isolated as controls.

F3-152

Accession no. X54156.

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.

Table 4.

PCR Analysis of Micromanipulated Single HRS Cells

Case HRS CellsB Cells Buffer Controls
Cells PCR Products CellsPCR Products Aliquots PCR Products
E4 E5/6 E7/8 E4 E5/6 E7/8 E4E5/6 E7/8
1  10  8  8  8  3  3  3  3  —  —  —  
2  10  8  8  8  1  1  1  3  —  —  —  
3  10  7  6  2  1  1  1  4  —  —  —  
4  12 11  9  8  10  4  4  4  15  1  1  
5  12  9  10  8  10  6  5  6  10  — —  —  
6  12  6  5  6  10  5  4  10  —  —  —  
7  12  4  4  4  10  5  4  10  —  —  —  
8  16  7  7  10  2  2  2  10  —  —  — 
Case HRS CellsB Cells Buffer Controls
Cells PCR Products CellsPCR Products Aliquots PCR Products
E4 E5/6 E7/8 E4 E5/6 E7/8 E4E5/6 E7/8
1  10  8  8  8  3  3  3  3  —  —  —  
2  10  8  8  8  1  1  1  3  —  —  —  
3  10  7  6  2  1  1  1  4  —  —  —  
4  12 11  9  8  10  4  4  4  15  1  1  
5  12  9  10  8  10  6  5  6  10  — —  —  
6  12  6  5  6  10  5  4  10  —  —  —  
7  12  4  4  4  10  5  4  10  —  —  —  
8  16  7  7  10  2  2  2  10  —  —  — 

Abbreviations: E4, exon 4 of the p53 gene; E5/6, exons 5 and 6; E7/8, exons 7 and 8.

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.

Table 5.

Coamplification of IgH Gene Rearrangements and Exons 4-8 of the p53 Gene From HRS Cells of Case 8

Cell5-150p53VH3
4 5/6 7/8
CD30+ 
  1  +  +  +  + (clonal) 
  2  +  +   + (clonal)  
  4  +   + (unique)  
  5  +    + (clonal)  
  6    + (clonal)  
  8  +  +  +  + (clonal) 
  9  +  +   + (clonal)  
 12  +  +  
 14   +  
CD20+ 
 36  +  +  
 43  +  +  
Cell5-150p53VH3
4 5/6 7/8
CD30+ 
  1  +  +  +  + (clonal) 
  2  +  +   + (clonal)  
  4  +   + (unique)  
  5  +    + (clonal)  
  6    + (clonal)  
  8  +  +  +  + (clonal) 
  9  +  +   + (clonal)  
 12  +  +  
 14   +  
CD20+ 
 36  +  +  
 43  +  +  
F5-150

HRS cells were micromanipulated as large CD30+ cells, control B cells as small CD20+ cells from immunostained sections of the HD biopsy specimen.

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 NFκB in the nuclei of HRS cells.25,26This might be caused by defective IκB or blocked expression of this inhibitor of NFκB.27 Constitutive nuclear expression of NFκB 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.38Our 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.

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.

REFERENCES

REFERENCES
1
Lukes
RJ
Butler
JJ
Hicks
EB
Natural history of Hodgkin’s disease as related to its pathological picture.
Cancer
19
1966
317
2
Kanzler
H
Küppers
R
Hansmann
ML
Rajewsky
K
Hodgkin and Reed-Sternberg cells in Hodgkin’s disease represent the outgrowth of a dominant tumor clone derived from (crippled) germinal center B cells.
J Exp Med
184
1996
1495
3
Küppers
R
Rajewsky
K
The origin of Hodgkin and Reed/Sternberg cells in Hodgkin’s disease.
Annu Rev Immunol
16
1998
471
4
Greenblatt
MS
Bennett
WP
Hollstein
M
Harris
CC
Mutations in the p53 tumor suppressor gene: Clues to cancer etiology and molecular pathogenesis.
Cancer Res
54
1994
4855
5
Takahashi
T
Nau
MM
Chiba
I
Birrer
MJ
Rosenberg
RK
Vinocour
M
Levitt
M
Pass
H
Gazdar
AF
Minna
JD
p53: A frequent target for genetic abnormalities in lung cancer.
Science
246
1989
491
6
Nigro
JM
Baker
SJ
Preisinger
AC
Jessup
JM
Hostetter
R
Cleary
K
Bigner
SH
Davidson
N
Baylin
S
Devilee
P
Glover
T
Collins
FS
Weston
A
Modali
R
Harris
CC
Vogelstein
B
Mutations in the p53 gene occur in diverse human tumour types.
Nature
342
1989
705
7
Imamura
J
Miyoshi
I
Koeffler
HP
p53 in hematologic malignancies.
Blood
84
1994
2412
8
Ichikawa
A
Kinoshita
T
Watanabe
T
Kato
H
Nagai
H
Tsushita
K
Saito
H
Hotta
T
Mutations of the p53 gene as a prognostic factor in aggressive B-cell lymphoma.
N Engl J Med
337
1997
529
9
Wilson
WH
Teruya-Feldstein
J
Fest
T
Harris
C
Steinberg
SM
Jaffe
ES
Raffeld
M
Relationship of p53, bcl-2, and tumor proliferation to clinical drug resistance in non-Hodgkin’s lymphomas.
Blood
89
1997
601
10
Gupta
RK
Patel
K
Bodmer
WF
Bodmer
JG
Mutation of p53 in primary biopsy material and cell lines from Hodgkin disease.
Proc Natl Acad Sci USA
90
1993
2817
11
Inghirami
G
Macri
L
Rosati
S
Zhu
BY
Yee
HT
Knowles
DM
The Reed-Sternberg cells of Hodgkin disease are clonal.
Proc Natl Acad Sci USA
91
1994
9842
12
Adamson
DJ
Thompson
WD
Dawson
AA
Bennett
B
Haites
NE
p53 mutation and expression in lymphoma.
Br J Cancer
72
1995
150
13
Xerri
L
Parc
P
Bouabdallah
R
Camerlo
J
Hassoun
J
PCRmismatch analysis of p53 gene mutation in Hodgkin’s disease.
J Pathol
175
1995
189
14
Chen
WG
Chen
YY
Kamel
OW
Koo
CH
Weiss
LM
p53 mutations in Hodgkin’s disease.
Lab Invest
75
1996
519
15
Elenitoba-Johnson
KS
Medeiros
LJ
Khorsand
J
King
TC
P53 expression in Reed-Sternberg cells does not correlate with gene mutations in Hodgkin’s disease.
Am J Clin Pathol
106
1996
728
16
Mansukhani
MM
Osborne
BM
Zhong
J
Matsushima
AY
The pattern of p53 and p21WAF1/CIP1 immunoreactivity in non-Hodgkin’s lymphomas predicts p53 gene status.
Diagn Mol Pathol
6
1997
222
17
Trümper
L
Daus
H
Roth
J
Loftin
U
Gause
A
Pfreundschuh
M
Single cell PCR at the DNA and RNA Level: A novel approach to the study of Hodgkin’s disease
Etiology of Hodgkin’s Disease.
Jarrett
R
1995
143
Plenum
New York, NY
18
Trümper
L
Brady
G
Bagg
A
Gray
D
Loke
SL
Griesser
H
Wagman
R
Braziel
R
Gascoyne
RD
Vicini
S
Iscove
NN
Cossman
J
Mak
TM
Single-cell analysis of Hodgkin and Reed-Sternberg cells: Molecular heterogeneity of gene expression and p53 mutations.
Blood
81
1993
3097
19
Jones
DB
Scott
CS
Wright
DH
Stein
H
Beverley
PC
Payne
SV
Crawford
DH
Phenotypic analysis of an established cell line derived from a patient with Hodgkin’s disease (HD).
Hematol Oncol
3
1985
133
20
Roers
A
Montesinos-Rongen
M
Hansmann
ML
Rajewsky
K
Küppers
R
Amplification of TCR beta gene rearrangements from micromanipulated single cells: T cells rosetting around Hodgkin and Reed-Sternberg cells in Hodgkin’s disease are polyclonal.
Eur J Immunol
28
1998
2424
21
Küppers
R
Zhao
M
Hansmann
ML
Rajewsky
K
Tracing B cell development in human germinal centres by molecular analysis of single cells picked from histological sections.
EMBO J
12
1993
4955
22
Lamb
P
Crawford
L
Characterization of the human p53 gene.
Mol Cell Biol
6
1986
1379
23
Rajewsky
K
Clonal selection and learning in the antibody system.
Nature
381
1996
751
24
Herbst
H
Epstein-Barr virus in Hodgkin’s disease.
Semin Cancer Biol
7
1996
183
25
Bargou
RC
Leng
C
Krappmann
D
Emmerich
F
Mapara
MY
Bommert
K
Royer
HD
Scheidereit
C
Dorken
B
High-level nuclear NF-kappa B and Oct-2 is a common feature of cultured Hodgkin/Reed-Sternberg cells.
Blood
87
1996
4340
26
Bargou
RC
Emmerich
F
Krappmann
D
Bommert
K
Mapara
MY
Arnold
W
Royer
HD
Grinstein
E
Greiner
A
Scheidereit
C
Dörken
B
Constitutive nuclear factor-kappaB-RelA activation is required for proliferation and survival of Hodgkin’s disease tumor cells.
J Clin Invest
100
1997
2961
27
Wood
KM
Roff
M
Hay
RT
Defective IkappaBalpha in Hodgkin cell lines with constitutively active NF-kappaB.
Oncogene
16
1998
2131
28
Levine
AJ
p53, the cellular gatekeeper for growth and division.
Cell
88
1997
323
29
Williams
GT
Smith
CA
Molecular regulation of apoptosis: Genetic controls on cell death.
Cell
74
1993
777
30
Drexler
HG
Recent results on the biology of Hodgkin and Reed-Sternberg cells. II. Continous cell lines.
Leuk Lymphoma
9
1993
1
31
Küppers
R
Rajewsky
K
Zhao
M
Simons
G
Laumann
R
Fischer
R
Hansmann
ML
Hodgkin disease: Hodgkin and Reed-Sternberg cells picked from histological sections show clonal immunoglobulin gene rearrangements and appear to be derived from B cells at various stages of development.
Proc Natl Acad Sci USA
91
1994
10962
32
Kanzler
H
Hansmann
ML
Kapp
U
Wolf
J
Diehl
V
Rajewsky
K
Küppers
R
Molecular single cell analysis demonstrates the derivation of a peripheral blood-derived cell line (L1236) from the Hodgkin/ Reed-Sternberg cells of a Hodgkin’s lymphoma patient.
Blood
87
1996
3429
33
Braeuninger
A
Küppers
R
Strickler
JG
Wacker
HH
Rajewsky
K
Hansmann
ML
Hodgkin and Reed-Sternberg cells in lymphocyte predominant Hodgkin disease represent clonal populations of germinal center-derived tumor B cells.
Proc Natl Acad Sci USA
94
1997
14211
34
Irsch
J
Nitsch
S
Hansmann
ML
Rajewsky
K
Tesch
H
Diehl
V
Jox
A
Küppers
R
Radbruch
A
Isolation of viable Hodgkin and Reed-Sternberg cells from Hodgkin disease tissues.
Proc Natl Acad Sci USA
95
1998
10117
35
Vockerodt
M
Soares
M
Kanzler
H
Küppers
R
Kube
D
Hansmann
ML
Diehl
V
Tesch
H
Detection of clonal Hodgkin and Reed-Sternberg cells with identical somatically mutated and rearranged VH genes in different biopsies in relapsed Hodgkin’s disease.
Blood
92
1998
2899
36
Brauninger
A
Hansmann
ML
Strickler
JG
Dummer
R
Burg
G
Rajewsky
K
Kuppers
R
Identification of common germinal-center B-cell precursors in two patients with both Hodgkin’s disease and non-Hodgkin’s lymphoma.
N Engl J Med
340
1999
1239
37
Finlay
CA
Hinds
PW
Tan
TH
Eliyahu
D
Oren
M
Levine
AJ
Activating mutations for transformation by p53 produce a gene product that forms an hsc70-p53 complex with an altered half-life.
Mol Cell Biol
8
1988
531
38
Pasman
PC
Tiebosch
A
Erdkamp
FL
Vrints
LW
Breed
WP
Schouten
HC
P53 as a marker of the malignant cell in Hodgkin’s disease.
Ann Oncol
5
1994
89
(suppl 1)

Author notes

Address reprint requests to Axel Roers, MD, Institute for Genetics, University of Cologne, Weyertal 121, 50931 Cologne, Germany.