FICTION-TSA analysis of the B-cell compartment in myeloma shows no signi®cant expansion of myeloma precursor cells FAITH E. DAVIES ,A NDREW C. R AWSTRON,G UY P RATT,S HEILA O'C ONNOR ,L ELA S U'UT,DAVID B LYTHE , J AMES F ENTON,DAVID C LAYDON, J. A NTHONY C HILD,A NDREW S. JACK AND G ARETH J. MORGAN Department of Haematology, The General In®rmary at Leeds, Leeds Received 6 January 1999; accepted for publication 31 March 1999 Summary. Studies utilizing ¯ow cytometry and PCR have shown that the B-cell compartment in myeloma contains cells which are clonally related to the myelomatous plasma cells. Current data, however, remains inconclusive regarding the extent of this involvement. By combining ¯uorescent immunophenotyping, tyramine signal ampli®cation and ¯uorescence in-situ hybridization (FICTION-TSA), we have used the presence of numerical chromosomal abnormalities within plasma cells as a clonal marker to examine the CD20  B-cell compartment for the presence of aneuploidy. A series of 54 cases of myeloma were screened for the presence of numerical abnormalities of chromosomes 3 and 11. FICTION-TSA was performed on 13 cases with either trisomy 3 or 11 and on a control group of six cases known to be disomic for the two chromosomes. B-cell numbers were reduced in the myeloma cases compared to the normal controls (median 1´8% v 3´0%, P  0´05). In the cases with a chromosomal marker, three signals were seen in a median of 1´88% of CD20  B cells compared to 2´58% within the control group. Comparison of the two groups using a Wilcoxon-Mann-Whitney U test showed no statistical signi®cant difference. Using this data set, it was possible to exclude a 3´03% expansion of clonally related B cells (95% con®dence level). We conclude that the B-cell compartment in myeloma does not represent the major site of clonal expansion, and if clonally related cells are present then the numbers are few. Keywords: multiple myeloma, ¯uorescence in-situ hybridization, FICTION, aneuploidy, B lymphocytes. Monoclonal gammopathy of uncertain signi®cance (MGUS) is assumed to be the precursor of clinically apparent myeloma, with one or more additional genetic events being required for the progression to myeloma. The patterns of mutation within the immunoglobulin heavy chain region provide a marker with which to study the relationship of these two conditions. In MGUS the malignant plasma cell population shows heterogeneity in the pattern of mutation, whereas in myeloma a homogenous pattern is seen (Bakkus et al, 1992; Sahota et al, 1996). One interpretation of this data is that in MGUS the progeny of an immortalized B cell continue to pass through a germinal centre before differ- entiating into plasma cells and migrating to the bone marrow. The homogenous mutation pattern seen in myeloma is thought to be the consequence of the expansion of a subclone of post-germinal centre cells. The exact site and cellular nature of the dividing population which leads to this clonal expansion has been the subject of much debate in recent years (Bakkus et al, 1994; Bergsagel et al, 1995; Billadeau et al, 1993; Chen & Epstein, 1996). Histological studies of reactive lymph nodes show a large number of post switch plasma cells within the germinal centre, suggesting that B cells may complete the process of plasma cell differentiation before migration to the bone marrow. However, in myeloma it has been suggested that a large number of B cells belonging to the myeloma clone are present in the peripheral blood and that these cells may represent a pool of precursor cells which migrate to the bone marrow and then mature into plasma cells (Bergsagel et al, 1995). One approach to determining where the clonal expansion occurs in myeloma is to compare the incidence of clonal genetic abnormalities within the B-cell and plasma cell populations. If the myeloma clone contains a substantial population of B cells, capable of proliferation and differentia- tion into plasma cells, it would be expected that the B cells would show the same range of genetic abnormalities as the plasma cells. British Journal of Haematology , 1999, 106, 40±46 40 q 1999 Blackwell Science Ltd Correspondence: Dr G. J. Morgan, Department of Haematology, Algernon Firth Building, The General In®rmary at Leeds, Great George Street, Leeds, West Yorkshire LS1 3EX.