Affiliations of authors: U. Hasse, M. F. Fey (Institute of Medical Oncology), E. O. Leibundgut, J.-F. Cajot, A. Tobler (Central Haematology Laboratory), Inselspital and Department of Clinical Research, University of Berne, Switzerland; M. Tinguely, B. Borisch, Department of Clinical Pathology, University of Geneva, Switzerland; A. M. Garvin, Department of Obstetrics and Gynaecology, Laboratory for Prenatal Medicine, University of Basel, Switzerland.
Correspondence to: Martin F. Fey, M.D., Institute of Medical Oncology, Inselspital, CH-3010 Berne, Switzerland (e-mail: martin.fey{at}insel.ch).
The typical Hodgkin and Reed-Sternberg (HRS) cells are thought to represent the malignant cellular elements of Hodgkin's disease (HD). The detection of immunoglobulin gene rearrangements in HRS cells indicates that they are clonally derived from B cells (1-8), but immunogenotyping, as such, does not provide any information on specific gene alterations possibly involved in the molecular pathology of HD. In many tumors, highly informative polymorphic DNA markers may identify loci harboring clonal loss of heterozygosity (LOH) and thus help to trace tumor suppressor genes (9,10). Although in HD, cytogenetic data suggest that nonrandom chromosomal deletions may occur at several loci, including 1q42, 4q26, and others, very little (if anything) is known about clonal LOH in HRS cells at the molecular level (11-15). We, therefore, set out to study microdissected HRS cells from classical types of HD at candidate loci for LOH with a highly sensitive microsatellite polymerase chain reaction (PCR) assay.
In seven patients with classical HD, HRS cells and surrounding cells including bystander
lymphocytes were laser microdissected from formalin-fixed, paraffin-embedded tissue sections
(Table 1). From a patient with nodular-sclerosing HD (patient 6), samples
were taken at presentation (6a) and at relapse (6b). In two patients (patient 6 and patient 7, a
patient with mixed cellularity-type HD), frozen sections of lymphoma tissue yielded
high-molecular-weight control DNA from microdissected cell populations. Buccal smears
provided constitutional high-molecular-weight DNA in patients 5 and 6 (16,17). Patients gave their informed consent to include their material in this study. For
molecular analyses, tetranucleotide repeat microsatellites were selected through the Genome Data
Base at loci with a high frequency of chromosomal deletions in HD, which were predicted by the
cytogenetic literature (11-15), and located at 1q42 (D1S517), 4q26
(D4S2301), 9p23 (D9S254), and 11q23 (D11S1294). Their PCR primer sequences are available
in the Genome Data Base (http://www.gdb.org). A highly sensitive
seminested PCR assay included a first round that used the primers indicated above. In a second
PCR, one of the primers in each pair was replaced by an internal forward primer (Fint) or an
internal reverse primer (Rint): 1q42 (D1S517Rint)
5'-CATGTGTCCATCAATGGTAG-3'; 4q26 (D4S2301Fint)
5'-GATGAGTGCTTAGACCATAGTA-3'; 9p23 (D9S254Rint)
5'-GTCTCCAATGCATGANCTT-3'; and 11q23 (D11S1294Rint)
5'-CTGGTTTGCTTTCCCTTTCTT-3' (software = "Primers! For
the WWW"; http://www.williamstone.com) (Fig. 1,
A). Allelic dropout during amplification of polymorphic microsatellite fragments may
randomly affect either allele mimicking LOH. To exclude this pitfall, pooled samples of 10
purified HRS cells from a given patient were amplified (18,19). Ten
picograms of DNA (corresponding to the DNA content of a single diploid cell) was consistently
detected by our PCR assay. We also used a seminested PCR (1,3,20-22)
to examine HRS cells from patients 3, 4, 6, and 7 for clonally rearranged immunoglobulin
heavy-chain genes created through joining of variable, diversity, and joining immunoglobulin gene
regions (V-D-J joining). The primers were a framework III immunoglobulin gene VH primer
5'-ACACGGC(C/T)(G/C)T(G/A)TATTACTGT-3' and a consensus JH primer (21) 5'-ACCTGAGGAGACGGTGACC-3'. The
seminested primer was from VLJH sequences
(5'-GTGACCAGGT(N)CCTTGGCCCCA-3') (22) (Fig. 1
, B).
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The detection of LOH has several implications in HD. The patients' buccal mucosa and bystander lymphocytes showed constitutional microsatellite patterns indicating that LOH in HRS cells was an acquired specific genetic feature of HD. To the best of our knowledge, this is the first report on microsatellite PCR detection of clonal LOH in microdissected HRS cells. In contrast to our approach, classical cytogenetics permit the study of mitotic cells only and usually do not permit the identification of the lineage of cells being karyotyped. The detection of LOH restricted to HRS cells is consistent with the view that they are clonally derived from a common single cell of origin (16,25-27). The probability in a given patient that all HRS cells tested would have lost the same allelic microsatellite band independently or by chance is .002 (i.e., 0.5n - 1, where n is the number of HRS cells examined) and, therefore, is low (28). Our findings thus confirm and extend molecular immunogenotype data on the clonality of HRS cells in classical HD with a molecular marker system unrelated to the configuration of immunoglobulin genes.
The detection of LOH in tumor cells at a particular locus indicates clonality and by the same token points to a site in the genome possibly harboring inactivated tumor suppressor genes. In non-Hodgkin's lymphomas, inactivation of tumor suppressor genes has been described at the p53 gene or the p16INK4a locus. Our work now shows that in classical HD, clonal LOH may be present at loci, such as 1q42, 4q26, 9p23, 11q22-23, and possibly others. The dense genomic map of microsatellite markers now available will help to narrow down such loci as a next important step for the eventual cloning of tumor suppressor genes operative in the molecular pathology of HD.
NOTES
Supported by grants 31-43458.95 and 3200-053596.98 (to A. Tobler and M. F. Fey) and grant 31.49681.96 (to B. Borisch) from the Swiss National Foundation; by grant KFS 156-9-1995 from the Swiss Cancer League; and by the Bernese Foundation for Clinical Cancer Research.
We thank Dr. Swee Lay Thein (John Radcliffe Hospital, Oxford, U.K.) for her helpful comments.
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Manuscript received March 16, 1999; revised July 1, 1999; accepted July 21, 1999.
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