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.
CD20-positive multiple myeloma – differential expression of cyclins D1 and D2 suggests a heterogeneous disease
Article first published online: 7 DEC 2009
© 2009 Blackwell Publishing Ltd
British Journal of Haematology
Volume 149, Issue 1, pages 156–159, April 2010
How to Cite
Quinn, J., Percy, L., Glassford, J., Somana, K., Rodriguez-Justo, M. and Yong, K. (2010), CD20-positive multiple myeloma – differential expression of cyclins D1 and D2 suggests a heterogeneous disease. British Journal of Haematology, 149: 156–159. doi: 10.1111/j.1365-2141.2009.08030.x
- Issue published online: 11 MAR 2010
- Article first published online: 7 DEC 2009
- Cyclin D;
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).
|Case||Age (years), sex||D-cyclin||Isotype||FISH||CD56||%CD20 expression||Distribution of CD20+ cells||Clinical course and outcome|
|1||49 f||D2||IgG λ||t(14;16)||neg||90||Diffuse||Dead – O.S. 25 months|
|2||44 m||D2||IgA κ||13qdel||N/A||90||Diffuse||Dead – O.S. 11·5 months|
|3||56 f||D2||IgA κ||NA||neg||50||Diffuse||Alive – O.S. 3 months|
|4||41 f||D2||κ– LC||Nil||neg||100||Diffuse||Alive – 10 RD, O.S – 49 months|
|5||70 f||D2||IgA κ||17pdel, 13qdel||neg||100||Diffuse||Dead – 10 RD O.S. – 0·3 months|
|6||75 m||D2||IgG κ||t(14;16)||neg||90||Diffuse||Alive – O.S. – 65·9 months|
|7||51 f||D2||IgG λ||13qdel||neg||100||Diffuse||Alive – 10 RD – O.S. – 17·2 months|
|8||45 f||D2||IgG λ||t(14;16)||N/A||30||Diffuse||Alive – O.S. – 11·1 months|
|9||58 f||D2||IgG λ||Nil||neg||70||Diffuse||Alive – O.S. – 14·5 months|
|10||72 m||D2||IgA κ||Nil||pos||20||Interstitial||Alive – 10 RD – 69·1 months|
|11||62 m||D2||IgG κ||N/A||neg||80||Clusters||Alive – O.S. – 17·9 months|
|12||59 m||D1||IgG κ||t(11;14)||neg||80||Diffuse||Alive – O.S – 85·3 months|
|13||57m||D1||IgG κ||Nil||neg||20||Interstitial||Alive – 10 RD – O.S. – 69·2 months|
|14||43 m||D1||IgM κ||Nil||neg||90||Diffuse||Dead – O.S. – 12·8 months|
|15||79 f||D1||IgG λ||Nil||pos||90||Interstitial||Alive – O.S. – 5 months|
|16||82 m||D1||λ– LC||13qdel||neg||90||Interstitial||Alive – 10 RD – O.S. – 43·8 months|
|17||62 f||D1||IgG κ||13qdel||neg||90||Interstitial||Alive – O.S. – 65·2 months|
|18||61 m||D1||IgA λ||t(11;14)||pos||50||Diffuse||Alive – O.S. – 79·5 months|
|19||58 f||D1||IgG λ||t(11;14)||pos||100||Diffuse||Alive – O.S. – 72·8 months|
|20||75 f||D1||IgG κ||Nil||pos||50||Diffuse||Alive – O.S. – 9·2 months|
|21||61 m||D1||IgD λ||t(11;14)||neg||100||Diffuse||Alive – O.S. – 5 months|
|22||73 f||N||IgG κ||N/A||pos||100||Interstitial||Alive – O.S. – 4 months|
|23||44 f||N||IgG λ||13qdel||pos||90||Interstitial||Dead – O.S. – 56·8 months|
|24||44 f||N||IgG κ||13qdel||NA||90||Interstitial||Alive – 30·3 months|
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%, n = 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.
This work was funded in part by a grant from Cancer Research UK.
- 2005) Cyclin D dysregulation: an early and unifying pathogenic event in multiple myeloma. Blood, 106, 296–303. , , , , & (
- 2005) Mutually exclusive cyclin-dependent kinase 4/cyclin D1 and cyclin-dependent kinase 6/cyclin D2 pairing inactivates retinoblastoma protein and promotes cell cycle dysregulation in multiple myeloma. Cancer Research, 65, 11345–11353. , , , , , , , & (
- 2008) Anti-CD20 monoclonal antibody therapy in multiple myeloma. British Journal of Haematology, 141, 135–148. , , , , & (
- 2005) Genetic abnormalities and patterns of antigenic expression in multiple myeloma. Clinical Cancer Research, 15, 3661–3667. , , , , , , , , , , , , , , , , & (
- 2008) Clonogenic multiple myeloma progenitors, stem cell properties, and drug resistance. Cancer Research, 68, 190–197. , , , , , , , , , , , & (
- 2002) Recurrent 14q32 translocations determine the prognosis of multiple myeloma, especially in patients receiving intensive chemotherapy. Blood, 100, 1579–1583. , , , , , , & (
- 2003) CD20 is associated with a small mature plasma cell morphology and t(11;14) in multiple myeloma. Blood, 102, 1070–1071. , , , , , , & (
- 2002) Clinicopathological and prognostic characteristics of CD56-negative multiple myeloma. British Journal of Haematology, 117, 882–885. , , , , , , , , , , & (
- 1991) Immunophenotypic heterogeneity of multiple myeloma: influence on the biology and clinical course of the disease. Castellano-Leones (Spain) Cooperative Group for the Study of Monoclonal Gammopathies. British Journal of Haematology, 1991, 77. , , , , , , , , & (
- 2006) The molecular classification of myeloma. Blood, 108, 2020–2028. , , , , , , , , , , , , , , , , , , & (