• CD20;
  • Cyclin D;
  • Myeloma

CD20 expression is found in approximately 15–20% of multiple myeloma (MM) patients, although the significance of such expression remains unclear (Kapoor et al, 2008). Overexpression of one of cyclins D1, D2 or D3 is ubiquitous in MM and several studies have shown an association between CD20 expression, cyclin D1 expression, and the t(11;14) translocation in particular. In one study, based on flow-cytometry of bone marrow aspirates, the t(11;14) translocation was found in 83% of CD20+ cases (10/12), a finding confirmed in a larger study in 2005 (Robillard et al, 2003; Mateo et al, 2005). Furthermore, immunohistochemical studies of MM bone marrow biopsies (Ely et al, 2005) and gene expression profiling experiments (Zhan et al, 2006) have also found an association between cyclin D1, t(11;14) and CD20 expression. Although an early study noted a poorer outcome for CD20+ MM cases (San Miguel et al, 1991), the t(11;14) translocation has subsequently been shown to be associated with a better prognosis (Moreau et al, 2002), thus the prognostic significance of CD20 expression in MM is unclear. In contrast to cyclin D1 expressing CD20+ MM cases, little is known about the biological or clinical behaviour of CD20+ MM cases expressing cyclin D2. Therefore, we aimed to characterise CD20+ MM cases associated with cyclin D2 expression by immunohistochemistry.

MM trephine biopsies where CD20 immunohistochemistry was performed were retrieved from departmental records and immunohistochemical findings were correlated with clinical data. CD20, cyclin D1 and cyclin D2 expression were determined by immunohistochemical staining of MM bone marrow biopsies. Samples were deemed to be positive for CD20 if >20% CD138+ cells in a biopsy had detectable expression of CD20. Intensity of CD20 staining and spatial distribution of CD20+ cells was also recorded. Fluorescence in-situ hybridization (FISH) analysis was performed using standard techniques.

Ninety-six cases were included (11 de novo MM, 13 relapsed) and CD20 expression was found in 24 (25%). Patient details are outlined in Table I. Ten cases expressed cyclin D1 and 11 cases expressed cyclin D2 by immunohistochemistry, confirmed in two cases by Western blotting (see Fig. 1A for representative sections). Three remaining CD20+ cases did not express cyclin D1 or D2. All 11 cyclin D2+ cases were entirely negative for cyclin D1. In the cyclin D2+ cases, the percentage of CD138+ cells expressing cyclin D2 was >50% in nine cases and between 25 and 50% in the remaining two cases. Our entire patient group (n = 96) included 12 patients with t(11;14) and CD20 expression was scored as positive in 4 of these 12 (33%), although CD20 expression was noted in 3 other cases with t(11;14) but in <20% of CD138+ cells. CD20 expression was seen in 3/5 cases with t(14;16), but none of eight cases with t(4;14). In the CD20+ cases, the median percentage of CD138+ cells in the trephine biopsies was 68% (range 20–100%) and the median percentage of CD20-expressing CD138+ cells was 90% (range 20–100%). CD20+ MM cells occurred in three patterns – diffuse (63%), interstitial (33%) and clustered (4%). Intensity of CD20 expression was moderate-to-strong in all cases (Fig. 1A).

Table I.   Patient details.
CaseAge (years), sexD-cyclinIsotypeFISHCD56%CD20 expressionDistribution of CD20+ cellsClinical course and outcome
  1. FISH, fluorescence in situ hybridization; f, female; m, male; NA, not available; N, no expression of cyclin D1 or D2; pos, positive; neg, negative; O.S, overall survival; 10 RD, primary refractory disease.

 149 fD2IgG λt(14;16)neg90DiffuseDead – O.S. 25 months
 244 mD2IgA κ13qdelN/A90DiffuseDead – O.S. 11·5 months
 356 fD2IgA κNAneg50DiffuseAlive – O.S. 3 months
 441 fD2κ– LCNilneg100DiffuseAlive – 10 RD, O.S – 49 months
 570 fD2IgA κ17pdel, 13qdelneg100DiffuseDead – 10 RD O.S. – 0·3 months
 675 mD2IgG κt(14;16)neg90DiffuseAlive – O.S. – 65·9 months
 751 fD2IgG λ13qdelneg100DiffuseAlive – 10 RD – O.S. – 17·2 months
 845 fD2IgG λt(14;16)N/A30DiffuseAlive – O.S. – 11·1 months
 958 fD2IgG λNilneg70DiffuseAlive – O.S. – 14·5 months
1072 mD2IgA κNilpos20InterstitialAlive – 10 RD – 69·1 months
1162 mD2IgG κN/Aneg80ClustersAlive – O.S. – 17·9 months
1259 mD1IgG κt(11;14)neg80DiffuseAlive – O.S – 85·3 months
1357mD1IgG κNilneg20InterstitialAlive – 10 RD – O.S. – 69·2 months
1443 mD1IgM κNilneg90DiffuseDead – O.S. – 12·8 months
1579 fD1IgG λNilpos90InterstitialAlive – O.S. – 5 months
1682 mD1λ– LC13qdelneg90InterstitialAlive – 10 RD – O.S. – 43·8 months
1762 fD1IgG κ13qdelneg90InterstitialAlive – O.S. – 65·2 months
1861 mD1IgA λt(11;14)pos50DiffuseAlive – O.S. – 79·5 months
1958 fD1IgG λt(11;14)pos100DiffuseAlive – O.S. – 72·8 months
2075 fD1IgG κNilpos50DiffuseAlive – O.S. – 9·2 months
2161 mD1IgD λt(11;14)neg100DiffuseAlive – O.S. – 5 months
2273 fNIgG κN/Apos100InterstitialAlive – O.S. – 4 months
2344 fNIgG λ13qdelpos90InterstitialDead – O.S. – 56·8 months
2444 fNIgG κ13qdelNA90InterstitialAlive – 30·3 months

Figure 1.  (A) Representative sections from three cases (1× cyclin D1+ and 2× cyclin D2+) showing CD138, CD20 and cyclin D expression. Two further haematoxylin & eosin (H + E) sections showing plasmablastic morphology in 2 cyclin D2+ cases along with Western blot showing cyclin D2 expression in CD138-selected cells from 2 cyclin D2+ cases. Immunohistochemical staining of tissue sections was performed on the Bond-maX system (Leica Biosystems, Newcastle, UK). Each case was stained with a panel of antibodies including: CD138 (Dako, dilution 1/50), CD20 (Dako, 1/400), CD56 (Novocastra, 1/00), Cyclin D1 (rabbit monoclonal antibody SP4, Lab Vision Products, 1/50) and Cyclin D2 (Cell Signalling Technology, cat no. 2924, at 1/50, using the Bond maX ER2 30′ antigen retrieval protocol). The cases were scored first by a haematopathologist and then by joint review on a multi-headed microscope. (B) Kaplan-Meier curves for cyclin D1 and D2 cases showing overall survival in months for both cyclin D1 and D2 groups.

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Although overall survival (OS) did not differ significantly between the two groups (Fig. 1B) there was a trend towards reduced OS in the D2 group (P = 0·29). In addition, primary refractory disease was more common in the D2 group (40%) in comparison with 20% in D1 group. Severe bone disease (i.e. with fractures) was present in 80% of D1+ group compared to just 27% in the D2 group. Plasmablastic morphology (27% vs. 0%) and lack of CD56-expression (86% vs. 64%, = 7 in each group) were also more common in the D2 group.

This study correlated CD20 expression in MM trephine biopsies with cyclin D2 protein expression by immunohistochemistry, and we believe this to be the first report of cyclin-D2 immunohistochemistry in MM trephine biopsies. We identified CD20 expression in 25% of 96 MM cases, a higher rate of expression than reported in studies involving flow-cytometric analysis of MM bone marrow aspirates (Mateo et al, 2005). We found that 43% (11/24) of the CD20+ MM cases expressed cyclin-D2 in keeping with the finding of overexpression of CCND2 mRNA in 40–50% of MM patients (Bergsagel et al, 2005). Despite this, most reports to date have emphasised the association between cyclin D1, CD20 expression and t(11;14). However, we found CD20 expression in just 33% of our 12 patients with the t(11;14) translocation. CD20 expression was not present in any of eight cases with t(4;14), but surprisingly was present in 60% (3/5) of cases with t(14;16).

In addition, we present data to suggest that CD20 expression in MM, when associated with cyclin D2 expression is an aggressive disease in some cases (whilst acknowledging the presence of poor prognostic factors in Cases 1 and 5). Although not statistically significant, in this small group of patients there was a trend towards shorter OS and higher rate of primary refractory disease in cyclin D2+ patients (Fig. 1B). Four patients in the D2 group and three in the D1 group have undergone autologous stem-cell transplantation. In the D2 group two patients relapsed at 6·8, and 12 months with the other two patients progression-free at 8 and 13 months. The three cyclin D1+ patients relapsed at 67, 78·1 and 73 months. Lack of CD56 expression was also more frequent in the D2 group, a feature associated with a more aggressive disease phenotype (Sahara et al, 2002).

A role for anti-CD20 therapy in MM has yet to be clearly established. Although single-agent rituximab produced only modest anti-myeloma activity in a Phase II study, reports exist of significant responses when used in combination with standard chemotherapy (Kapoor et al, 2008). Furthermore, CD20 remains an appealing target in MM, with the possibility of MM ‘progenitor’ cells expressing CD20 (Matsui et al, 2008). In conclusion, we present data showing that CD20+ MM is a heterogeneous entity, with a cyclin D2+ sub-group displaying more aggressive disease. If confirmed in a larger analysis, perhaps patients with cyclin D2-positive, CD20+ MM should be considered for anti-CD20 therapy, particularly if poor prognostic features are present.


  1. Top of page
  2. Acknowledgements
  3. References

This work was funded in part by a grant from Cancer Research UK.


  1. Top of page
  2. Acknowledgements
  3. References
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