Clonal Loss of Heterozygosity in Microdissected Hodgkin and Reed-Sternberg Cells
Authors: U. Hasse, M. Tinguely, E. O. Leibundgut, J.-F. Cajot, A. M. Garvin, A. Tobler, B. Borisch, M. F. Fey
Journal: JNCI Journal of the National Cancer Institute (1999)

The typical Hodgkin and ReedSternberg (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 nodularsclerosing 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 cellularitytype 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 (Rin t ) : 1q42 (D1S517Rin t ) 5 8 CATGTGTCCATCAATGGTAG-38; 4q26 (D4S2301Fint) 58-GATGAGTGCTTAGACCATAGTA-38 ; 9p23 (D9S254Rint) 58-GTCTCCAATGCATGANCTT-38; and 11q23 (D11S1294Rint) 58-CTGGTTTGCTTTCCCTTTCTT-38 (software 4 “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 58ACACGGC(C/T)(G/C)T(G/A)TATTACTGT-38 and a consensus JH primer (21) 58-ACCTGAGGAGACGGTGACC-38. The seminested primer was from VLJH sequences (58-GTGACCAGGT(N)CCTTGGCCCCA-38) (22) (Fig. 1, B). DNA extracted from buccal smears and from bystander lymphocytes showed constitutional individualspecific microsatellite patterns in all patients (Fig. 1, A). Of 259 samples of HRS cells microdissected from formalin-fixed tissue, 59 (23%) yielded a detectable microsatellite PCR product. Mock picks from tissue sections cleared of cellular material were consistently negative by PCR analysis. All patients except patient 5 reproducibly showed clonal LOH in HRS cells at one to three different microsatellite loci (Table 1). LOH was seen at all loci, but the locus most frequently altered was D4S2301 (4q26; four of five informative patients). High-molecular-weight DNA from fresh HRS cells and corresponding degraded DNA from formalin-fixed tissue yielded identical individual-specific microsatellite band patterns (Fig. 1, A). None of the HRS cell samples showed extra microsatellite bands (23). Patients 3, 4, 6, and 7 showed clonal immunoglobulin heavy-chain gene configurations in HRS cells and polyclonal patterns in bystander lymphoid cells (Fig. 1, B). In patient 6, a common rearranged immunoglobulin heavy-chain gene band in HRS cells, observed at presentation (6a) and at relapse (6b), indicated that the initial monoclonal population of HRS cells had relapsed. Our immunogenotype data from formalin-fixed HRS cells are in keeping with published data that HRS cells are mostly monoclonal B-cell populations (1–8,20,24). 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 identifi-