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Multiple Myeloma |
From the Department of Immunology, St. Helier Hospital, Carshalton, Surrey, UK (MJA); Section of Haemato-Oncology, The Royal Marsden NHS Foundation Trust, UK (FED, DdC, GJM)
Correspondence: Michael J. Ayliffe, Department of Immunology, St Helier Hospital Wrythe Lane, Carshalton, Surrey, SM3 8NR, UK. E-mail: michael.ayliffe{at}onetel.net
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ABSTRACT |
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 TOP ABSTRACT Design and MethodsResults and DiscussionReferences |
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Key words: multiple myeloma, bone marrow, plasma cells, free light chains, monoclonal immunoglobulins.
Multiple myeloma (MM) is characterized by a malignant proliferation of monoclonal plasma cells in the bone marrow and of their products in serum and/or urine. M-Igs and FLCs are used to monitor disease and treatment. FLCs are found in the urine of 68% of MM cases. So-called Bence-Jones proteinuria (BJP) and increases of FLCs in serum are associated with disease progression.1,2 Up to 20% of MM cases have FLC only disease in which light chains in the serum and/or urine is the only immunochemical abnormality found.3 The serum assay for FLCs in MM has been extensively investigated.4 A shift to the secretion of light chains has been associated with relapse but little cellular information is available derived from direct visualisation of light chain production.
Immunofluorescence of cytocentrifuge preparations of marrow cells is a useful and reproducible approach to define the proportion of plasma cells that are monoclonal.5 Double staining is preferable to single staining as each population is enumerated separately. It also has the advantage of an internal control. Since plasma cells only contain Igs of a single heavy chain and light chain type, the sum of the percentages of the heavy chain figures should approximate to 100%, This is also true of the sum of the light chain percentages. Discrepancies in these figures led to the identification in individual cases of separate populations of cells staining for either intact M-Igs or FLCs only. These dual bone marrow populations form the subject of this brief report.
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Design and Methods |
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TOPABSTRACTDesign and MethodsResults and DiscussionReferences |
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light chain only 20/146 (14%); 
light chain only 11/146 (8%); biclonal gammopathies 8/146 (6%); and non-secretory MM 2/146 (1%). The male to female ratio was 2:1 and the median age at presentation was 53 (range 28–79). In addition, 38 cases of Waldenstrom’s macroglobulinemia, 28 cases with monoclonal gammopathy of undetermined significance (MGUS), 17 with solitary or extramedullary plasmacytomas, 18 with AL amyloidosis, 10 with nonsecretory MM and 12 with plasma cell leukemia were reviewed for the presence of dual populations.
Cytocentrifuge preparations were made as previously described. 5 Eight preparations were fixed and stained in separate mixtures of anti-Ig reagents as follows: monospecific antisera to human IgG, IgM, IgA and IgD heavy chains and to and light chains conjugated to fluorescein isothiocyanate (FITC) were each mixed with poly-specific anti-human Ig serum conjugated to rhodamine isothiocyanate (TRITC), (Dako). Specimens containing dual populations were confirmed by restaining with anti-heavy chain FITC and anti-light chain TRITC reagents of appropriate isotype (Figure 1).
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M-Igs in the serum and BJP in the urine were detected by electrophoresis and identified by immunofixation using monospecific anti-Ig reagents and gold stain. Abnormal bands were quantified by densitometric scanning.
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Results and Discussion |
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TOPABSTRACTDesign and MethodsResults and DiscussionReferences |
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Intact M-Igs were detected in the sera of 113/146 (78%) of these MM cases. The staining of the predominant marrow plasma cells corresponded with the heavy and light chain of the M-Igs detected in the serum in all but one case. This patient’s cells stained as a biclonal IgG and IgA but only monoclonal IgA was detected in the serum. In 7/113 (6%), the sum of the heavy chain staining cells was substantially lower than that of the light chain staining cells, indicating a subpopulation restricted to light chain staining only. BJP was detected in the urine of 6/7 (86%) of these MIg+ dual population cases.
FLC only disease was diagnosed in 31/146 (22%) of cases. A subpopulation of marrow cells containing monoclonal heavy chains was detected in 11/31 (35%) of these cases (Table 1). Many dual population samples gave a characteristic pattern of dim and bright staining with the anti-Ig TRITC reagent. This may be due to the M-Ig+ cells binding to two components (anti-heavy and anti-light chain) of the pentavalent antiserum while the FLC restricted cells bind to only one.
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Similar results were found in other plasma cell dyscrasias. Dual populations were found in 1/28 (4%) of MGUS cases and in 3/18 (17%) of cases with AL amyloidosis but none in 38 cases of Waldenstrom’s macroglobulinemia. Monoclonal plasma cell populations were found in 7/19 (37%) of investigations of solitary or extramedullary plasmacytomas and one of these had a dual population. No dual populations were seen in 10 cases with non-secretory MM. All had a monoclonal plasma cell population and 5 of them showed single populations of FLC only cells. In 12 cases of plasma cell leukaemia there were no dual populations but 5/12 (42%) had a single population of FLC only cells.
The double immuno-staining technique employed here allowed the direct demonstration of the emergence of dual plasma cell subsets in MM that have been suggested for many years by the association of disease progression originally with BJP and more recently increases in serum FLCs. Single-staining or the and staining which are frequently used to establish monoclonality would not detect these differences. The presence of dual populations in a proportion of both intact M-Ig+ and FLC only cases and the 12% who changed their cellular protein profile suggest that such changes in subsets are not uncommon. Although VL sequences were not available to prove that the two populations were clonally related in individual cases this is highly likely since intraclonal heterogeneity is not known in MM. Loss of heavy chains is likely to be the result of acquired genetic aberrations in the functional VDJH alleles at the genomic DNA level. However, mechanisms acting at the mRNA level cannot be excluded.
We showed BrDU labeling of both populations (Figure 1) but only 1 case was associated with morphological differences.6,7 Tumor escape mutants may be expected to increase with modern treatment and longer patient survival and testing for M-Ig and FLC will remain imperative.8 These population changes may contribute to a cellular basis for some disease progression, for changes in serum FLC concentrations, for socalled Bence-Jones escape9 and the FLC breakthrough that has been recently reported.10,11 The proliferative advantage, and the molecular, genetic and drug sensitivity cellular mechanisms influencing the natural history of MM could be further investigated if the two component populations were separated by flow cytometry.
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Footnotes |
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MA conceived and designed the study, acquired, analysed and interpreted the data and was involved in the drafting, revision and decision to submit the paper for publication; FD interpreted data, provided clinical input and was involved in the drafting, revision and the decision to submit the paper for publication; DG interpreted data, provided genomic input and was involved in the drafting, revision and the decision to submit the paper for publication; GM interpreted data, provided clinical input and was involved in the drafting, revision and the decision to submit the paper for publication.
The authors reported no potential conflicts of interest.
Received for publication March 22, 2007. Accepted for publication May 26, 2007.
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References |
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TOPABSTRACTDesign and MethodsResults and DiscussionReferences |
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