• cyclin D1;
  • t(11;14);
  • multiple myeloma;
  • BrdU LI;
  • E2F-1


  1. Top of page
  2. Abstract
  3. Patients and methods
  4. Results
  5. Discussion
  6. References

Cyclin D1, encoded by the CCND1 gene, is immunohistochemically detectable in up to one-third of cases of multiple myeloma (MM). To examine the mechanism of cyclin D1 overexpression, we compared cyclin D1 immunoreactivity with the results of conventional cytogenetics to determine if the t(11;14)(q13;q32) or other abnormalities of 11q11–14 explained cyclin D1 overexpression. Karyotypic abnormalities were found in 45 out of 67 (67%) MM cases; the t(11;14) was present in seven cases (10%). Additional 11q11–14 abnormalities were not identified. The t(11;14) correlated with cyclin D1 upregulation in low to intermediately proliferative MM, but was not present in highly proliferative tumours (assessed using bromodeoxyuridine labelling index). Cyclin D1 indirectly activates the transcription factor E2F-1. In the bone marrow biopsy specimens of MM cases, E2F-1 was concurrently expressed with cyclin D1 (P = 0·001), indicating that cyclin D1 is functional. However, as neither E2F-1 nor cyclin D1 expression correlated with proliferative activity, the speculation that t(11;14) upregulates the CCND1 gene to induce higher proliferation and possibly more aggressive disease is not supported. We conclude that in low to intermediately proliferative MM cases, cyclin D1 is probably upregulated by t(11;14), but an alternative mechanism is more probable in highly proliferative MM.

Defects of normal cell cycle regulation, particularly at the G1 to S phase transition, play a pivotal role in tumour development (Sherr, 1996; Reed, 1997). Cyclin D1 is one of the most important of the many activating and inhibitory proteins that affect G1 to S phase progression (Hunter & Pines, 1994). Cyclin D1 activates cyclin-dependent kinases (CDKs) to trigger phosphorylation of retinoblastoma protein (Weinberg, 1995). Phosphorylation causes release of the transcription factor E2F-1, which is normally bound to retinoblastoma protein (Chellappan et al, 1991). E2F-1, in its free and active form, is thought to be the ultimate factor that promotes transition into S phase and mitosis (Johnson et al, 1993).

Cyclin D1 protein is overexpressed in mantle cell lymphoma as a result of the t(11;14) translocation, in which the CCND1 (also known as PRAD1) gene on chromosome 11q13 is juxtaposed with the immunoglobulin heavy chain gene at 14q32 (Seto et al, 1992). Deregulated expression of cyclin D1 results in these cells having a proliferative advantage. In multiple myeloma (MM), the t(11;14) is identified in approximately 4–10% of bone marrow (BM) specimens when examined using conventional cytogenetics and in 12–16% of BM specimens assessed using fluorescent in situ hybridization (FISH) methods (Lai et al, 1995; Sawyer et al, 1995; Nishida et al, 1997; Avet-Loiseau et al, 1998). In contrast, cyclin D1 protein is immunohistochemically detectable in up to one-third of MM cases and may be associated with greater tumour burden (Vasef et al, 1997; Lai et al, 1998). The relationship between cyclin D1 expression and t(11;14) is unclear in these cases (Kobayashi et al, 1995). If cyclin D1 is functional and involved in the pathogenesis of MM, cytogenetic studies are either insufficiently sensitive to detect the t(11;14), or cyclin D1 expression occurs independently of t(11;14) and may exert its activity in collaboration with other oncogene products, as occurs in many other tumour types (Motokura & Arnold, 1993).

In this study, we attempt to address three questions. First, what is the mechanism of cyclin D1 expression in MM and, in particular, can it be explained by cytogenetic abnormalities? To answer this question, we examined the relationship between cyclin D1 protein expression and t(11;14) or other chromosome 11q abnormalities detectable using cytogenetic studies in BM specimens from MM patients. Second, is cyclin D1 functional in MM? As cyclin D1 overexpression should result in increased free E2F-1 protein, we assessed the cases immunohistochemically with an antibody specific for E2F-1. Finally, to determine if cyclin D1 overexpression results in increased tumour proliferation, either independently or by activating E2F-1, trephine biopsy sections of MM with predetermined bromodeoxyuridine labelling index (BrdU LI) were selected for evaluation of both cyclin D1 and E2F-1 immunostaining. The BrdU LI determines the percentage of plasma cells in S phase and is used in plasma cell dyscrasias to estimate the proliferative activity of BM plasma cells (Ffrench et al, 1995; Joshua et al, 1996).

Patients and methods

  1. Top of page
  2. Abstract
  3. Patients and methods
  4. Results
  5. Discussion
  6. References

Case selection The study population included all MM patients with BM specimens submitted for analysis of proliferative activity, using the BrdU LI technique, between May 1996 and December 1997 at the University of Arkansas for Medical Sciences, Little Rock, AR, USA. The first BrdU LI for each patient in this time period was recorded. Seventy-five out of 364 patients with a positive BrdU LI of ≥ 0·2 were selected for evaluation using the following method. The first five MM patients at stratified BrdU levels outlined in Fig 1 (essentially, 0·2 intervals from 0·2 to 2·0, and 1·0 intervals between 2·0 and 7·0) who had complete data in the database and > 30% BM plasma cells were identified. Among the initial 75 cases, 11 were rejected owing to insufficient material remaining in BM biopsy paraffin blocks (10 cases) or absence of the paraffin block (one case). The rejected cases were replaced by the next patient in their category, except for two patients with a BrdU LI of 5·0–5·9 and 6·0–6·9, who had no replacements. In addition, one case was removed after it was discovered that a name change resulted in one patient being evaluated twice (BrdU LI of 5·0–5·9). The final sample size for evaluation was 72 patients.


Figure 1. Percentage of MM cases evaluated at each BrdU LI. Fifteen levels of BrdU LI were selected for examination as follows: 0·2 intervals from BrdU LI 0·2–2·0, 1·0 intervals between BrdU LI 2·0 and 7·0, and cases with BrdU LI ≥ 7·0. Five patients were examined at each level from a total of 364 patients as described in Patients and Methods.

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Bromodeoxyuridine (BrdU) labelling index Mononuclear cells were isolated from BM aspirate specimens using Ficoll-Hypaque (Sigma, St. Louis, MO, USA) gradient separation and incubated for 1 h at 37°C in media containing 10% fetal bovine serum, 40 μmol/l 5-bromo-2′-deoxyuridine and 5-fluoro-2′-deoxyuridine (Sigma). After culture, cells were extensively washed, cytocentrifuged onto glass slides, fixed in 70% ethanol and treated with 4 mol/l HCl for DNA strand separation. Cells were stained with rhodamine-conjugated rabbit anti-human kappa and lambda antibodies (Dako, Carpinteria, CA, USA). BrdU uptake was identified using a mouse anti-BrdU monoclonal antibody (IgG1, Becton-Dickinson, San Jose, CA, USA) followed by fluorescein-conjugated goat anti-mouse IgG1-specific antibodies (Southern Biotechnology Associates (Birmingham, AL, USA). Five hundred kappa- or lambda-positive plasma cells were counted using fluorescent microscopy and the number of BrdU-positive plasma cells was determined and expressed as a percentage (labelling index) (Greipp et al, 1987).

Immunohistochemical studies Zenker-fixed, paraffin-embedded BM trephine biopsy sections were routinely processed and mounted onto ChemMate capillary gap slides (Ventana Biotek Medical Systems, Tucson, AZ, USA). Immunostaining was performed using an automated immunostainer (TechMate 1000; Ventana Biotek Medical Systems) and the following antibodies: E2F-1 (clone KH95, 1:50, Santa Cruz Biotechnology, Santa Cruz, CA, USA); a cocktail of two cyclin D1 antibodies (clone 2D11F11, 1:20, Novocastra Laboratories/Vector Laboratories, Burlingame, CA, USA, combined with clone 5D4, 1:50, Immunotech, Westbrock, ME, USA); and Ki-67 (MIB-1, 1:100, Immunotech). All sections were placed in 0·01 mol/l citrate buffer at pH 6·0 (HIER Buffer, Ventana Biotek Medical Systems) and heated twice in a microwave (5 min/cycle) prior to staining. In addition, sections for cyclin D1 immunodetection underwent sonication-induced epitope retrieval as previously described (Brynes et al, 1997). Reactivity was detected with an avidin–biotin immunoperoxidase detection system employing 3′, 3′-diaminobenzidine-tetrahydrochloride dihydrate (Ventana Biotek) as the chromogen. Sections of multitissue mantle cell lymphoma and reactive tonsil control blocks served as positive and negative controls.

To evaluate staining, sections were scanned at low power to identify areas that were evenly labelled with cyclin D1, E2F-1 or Ki-67. Cyclin D1 and E2F-1 were considered positive if nuclear staining was present in > 5% of plasma cells at 400× magnification in any area of the tumour (Lai et al, 1998). Nuclear staining of endothelial cells served as an internal control. Ki-67 positivity was graded as the percentage of total plasma cells with positive nuclear staining, either < 10%, 10–20% or > 20% plasma cells. As previously described (Lai et al, 1998), this corresponded to low, intermediate and high proliferative rates. All cases were evaluated independently by three authors without knowledge of the patient's BrdU LI, cytogenetic or clinical status. Of 216 slides evaluated, the three observers jointly reviewed 31 slides (14%) at a multiheaded microscope and discrepancies or clarifications in staining on these cases were reconciled. In addition, it was determined that one slide (E2F-1 stain) could not be evaluated.

Morphology Haematoxylin and eosin-stained trephine biopsy sections were reviewed for the histological type of MM based on the Bartl et al (1987) classification as follows: grade I – low grade (marschalko and small cell types), grade II – intermediate grade (cleaved, polymorphous and asynchronous types), grade III – high grade (blastic type).

Cytogenetic studies Bone marrow specimens underwent cytogenetic analysis using standard techniques as previously described (Sawyer et al, 1995). A direct collection, and 24- and 48-h cultures were obtained. Examination of 20 metaphases was attempted on all specimens. The presence of two metaphases with the same structural abnormality or the same extra chromosome, or three metaphases with the same missing chromosome, defined a clonal abnormality. Aberrations were designated according to the International System for Human Cytogenetic Nomenclature (ISCN, 1995).

Flow cytometry studies Bivariate flow analysis of cytoplasmic Ig light chains and DNA content was performed on BM aspirate material using a modification of a technique published previously (Barlogie et al, 1985). Ficoll-Hypaque-separated cells were exposed to either Ig kappa or lambda light chain reagents (fluorescein-conjugate F(ab′)2 fragment, Dako) and counterstained for DNA with propidium iodide (Sigma). Proportions of tumour cells were determined by applying an electronic gating procedure to analyse the brightly Ig light chain-positive cells with abnormal DNA content (aneuploid population) or the relative excess of Ig kappa or lambda light chain-positive cells with normal DNA content (diploid population). Routinely, 5000 cells were analysed with a FACScan instrument (Becton Dickinson).

Statistical analysis Statistical analysis was performed using SigmaStat for Windows 2·0 software (Jandel, San Rafael, CA, USA). Comparison of variables involving non-parametric data was determined using the Spearman Correlation test. Fisher's exact test was used to compare differences in a variable between groups. Differences were considered significant at P < 0·05.


  1. Top of page
  2. Abstract
  3. Patients and methods
  4. Results
  5. Discussion
  6. References

Patient characteristics

The age of the 72 patients ranged from 27 to 77 years (median, 57 years). There were 40 men (56%) and 32 women (44%). One MM case was non-secretory and the remaining cases synthesized the following paraproteins: 32 IgG kappa, 11 IgG lambda, 11 IgA kappa, seven IgA lambda, seven kappa light chain and three lambda light chain.

Proliferative activity

MM cases were selected based on their BrdU LI, with five cases of MM examined at each BrdU LI interval, as previously described. Figure 1 shows the BrdU LI of the MM studied compared with the entire population of 364 patients. The study design selected for a disproportionate number of cases with intermediate to high BrdU LI, as the majority of the group had a BrdU LI < 2·0. For example, only 9% of our MM cases with a BrdU LI of 0·2 were examined compared with 100% of MM cases with a BrdU LI of 6·0–6·8. BrdU LI in BM aspirate specimens strongly correlated with Ki-67 immunostaining in the concurrent BM trephine biopsy sections (P < 0·0001). Increased proliferative activity also correlated with worsening cytological differentiation or higher Bartl grade (BrdU LI, P = 0·02; and Ki-67, P = 0·001).

Immunohistochemical results

Cyclin D1 was expressed in 22 (31%) MM cases (Fig 2) and E2F-1 was expressed in 25 (35%) MM cases. E2F-1 positivity showed a significant correlation with cyclin D1 expression (P < 0·001, Table I). A significant relationship between E2F-1 or cyclin D1 reactivity and proliferative rate was not found, measured by either BrdU LI or Ki-67 positivity. However, women had cyclin D1-positive MM (16 out of 32) (P = 0·028) more often than men (6 out of 40) and, in particular, women with cyclin-D1-positive MM of IgA isotype had a disproportionate number of tumours with high proliferative activity. Among patients with IgA-positive MM, seven out of nine (78%) women compared with one out of eight (12%) men (P = 0·015) had cyclin D1-positive MM.


Figure 2. Nuclear staining of cyclin D1 in a case of MM (immunoperoxidase, 500x original magnification).

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Table I.  Frequency of E2F-1 immunoreactivity, karyotypic abnormalities and aneuploid populations in MM as stratified by BrdU LI.
 BrdU labelling index 
 < 1·01·0–2·8≥ 3·0Total
  • *

    E2F-1 staining in one case could not be evaluated.   

  • Fisher's exact test, P < 0·001.   

  • ‡Five cases not included owing to insufficient metaphases for cytogenetic evaluation.

E2F-1 positive
 Cyclin D1+5/6 (83%)7/10 (70%)4/5 (80%)*16/21 (76%)
 Cyclin D1–1/14 (7%)5/20 (25%)3/16 (19%)9/50 (18%)
 Abnormal karyotype5/17 (29%)18/28 (64%)‡22/22 (100%)45/67 (67%)
Flow cytometry
 Aneuploid population16/20 (80%)23/30 (77%)17/22 (77%)56/72 (78%)

Cytogenetic studies

Cytogenetic results were obtained in 67 (93%) cases (Table I). The five cases with inadequate metaphases included three cases with BrdU LI < 1·0 (cyclin D1 negative) and two cases with BrdU LI 1·0–1·8 (cyclin D1 positive). The presence of an abnormal karyotype was associated with higher proliferative activity. An abnormal karyotype was found in 75% (15 out of 20) of cyclin D1-positive cases, 64% (30 out of 47) of cyclin D1-negative cases and 67% (45 out of 67) of all cases. The t(11;14) was present in 35% (7 out of 20) of the cyclin D1-positive cases, was absent in all cyclin D1-negative cases and was present in 10% (7 out of 67) of cases overall (Fig 3). The translocation was seen only in cyclin D1-positive, low to intermediately proliferative MM cases (BrdU LI < 3·0). The incidence of t(11;14) among MM with abnormal karyotypes in this group was 79% (seven out of nine). In addition, one MM in this group with insufficient material for cytogenetic evaluation was shown to carry the t(11;14) in a subsequent BM evaluation 12 months later.


Figure 3. Percentage of (A) cyclin D1-positive and (B) cyclin D1-negative MM with abnormal cytogenetic karyotypes stratified by BrdU LI. Cases with t(11;14) are represented by white bars. Cases with other cytogenetic abnormalities and no t(11;14) are represented by black bars.

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Six out of 22 cases with highly proliferative MM were cyclin-D1 positive. Of these, 67% (four out of six cases) were from women with IgA-secreting MM. All highly proliferative MM (BrdU LI ≥ 3) had complex cytogenetic abnormalities and none had the t(11;14). A high incidence of monosomy 13 or 13q deletions (64%, 14 out of 22 cases) and chromosomal translocations (82%, 18 out of 22 cases) was present, independent of cyclin D1 status. Chromosome 11 abnormalities consisted predominately of trisomy 11 (33%, 5 out of 15 cyclin D1-negative cases vs. 29%, 2 out of 7 cyclin D1-positive cases). No evidence of 11q13 abnormalities or variant t(11;14) translocations was identified in any of the cyclin D1-positive cases.

Flow cytometry

Aneuploid plasma cell populations were present in 56 (78%) cases and, unlike cytogenetic abnormalities, were seen uniformly among tumours with differing BrdU LI levels (Table I). In particular, 80% of low proliferative MM (BrdU LI < 1·0) had aneuploid cell populations despite the majority of normal karyotypes (71%).


  1. Top of page
  2. Abstract
  3. Patients and methods
  4. Results
  5. Discussion
  6. References

Cyclin D1 immunoreactivity was identified in 31% of BM biopsy specimens of MM, a frequency similar to previous studies (Vasef et al, 1997; Lai et al, 1998). Abnormal karyotypic abnormalities were found in 67% of cases and were related to the proliferative activity of the tumours. All highly proliferative MM cases, as measured by BrdU LI, had cytogenetic abnormalities, compared with less than 30% of cases with low proliferative disease. The t(11;14), detected in 10% of all BMs, was found exclusively in cyclin D1-positive cases. Among low to intermediately proliferative MM with abnormal karyotypes, 79% of cyclin D1-positive but no cyclin D1-negative tumours carried the t(11;14), suggesting a significant relationship. The t(11;14) was probably underrepresented using cytogenetic analysis in this population, given the presence of aneuploid populations in 80% of low proliferative MM determined by flow cytometric evaluation.

The t(11;14) was not detected in MM with high proliferative activity, despite the finding of cyclin D1 positivity in 37% of cases. Other abnormalities of 11q11–14 that could account for overexpression of CCND1 were also not identified. While clonal plasma cell subpopulations with t(11;14) may have been missed (Hallek et al, 1998), attempts to better define genetic defects in MM with multicolour spectral karyotyping (SKY) and FISH analysis do not support this possibility. We performed SKY on 50 MM cases with complex chromosomal aberrations that were not fully characterized by G-banding, but had no evidence of t(11;14) (Sawyer et al, 1998). The t(11;14) was identified in only one case, involving 14q21 rather than the 14q32. Avet-Loiseau et al (1999) used metaphase FISH analysis to identify t(11;14) in only one of 32 MM negative for the t(11;14) using G-banding techniques. Similarly, Nishida et al (1997) applying dual-colour FISH on metaphase spreads and interphase nuclei, detected the t(11;14) in 5 out of 47 (10%) plasma cell malignancies, four of which had normal G-banded karyotypes. The single patient with an abnormal (complex) karyotype and detection of t(11;14) using FISH had plasma cell leukaemia. Extrapolation of these findings suggests that t(11;14) may be missed by cytogenetic studies in low to intermediately proliferative MM with normal karyotypes, but adequate detection of t(11;14) usually occurs in MM with abnormal karyotypes. Possible alternative mechanisms for cyclin D1 expression in the highly proliferative tumours include abnormalities in CDK inhibitors that normally compete with cyclin D1 for binding to CDKs (Kawano et al, 1997; Ng et al, 1997; Tasaka et al, 1997; Urashima et al, 1997) and truncation with increased stabilization of CCND1 mRNA (Hirama & Koeffler, 1995). Regardless of the exact mechanism, cyclin D1 in highly proliferative MM may act as a protooncogene in concert with other oncogenes to transform cells (Motokura & Arnold, 1993; Hunter & Pines, 1994).

The t(11;14) has been suggested to be a poor prognostic indicator in some studies of patients with MM (Fonseca et al, 1999), but in other studies it had no prognostic significance (Avet-Loiseau et al, 1998; Sonoki et al, 1999). In this study, the cyclin D1 protein appeared to be functionally active because expression strongly correlated with E2F-1 immunoreactivity. However, a significant relationship between cyclin D1 or E2F-1 expression and increased proliferative activity of MM was not found, despite the role cyclin D1 and E2F-1 play in promoting cells from G1 to S phase of the cell cycle. Evaluating proliferative activity using the BrdU LI, which measures the percentage of plasma cells in S phase in BM aspirate material (BrdU LI), correlated strongly with Ki-67 immunostaining of BM biopsy sections, which discriminates between cycling (G1, S and G2 + M) and resting (G0) plasma cells (Falini et al, 1988; Tsurusawa et al, 1995). Therefore, factors other than cyclin D1 and E2F-1 must be affecting these tumours. For example, variations in cycle times in the heterogenous plasma cell populations (Shackney & Shankey et al 1999), or a second oncogene, similar to the putative transforming gene myeov found in a subset of MM cell lines with t(11;14) (Janssen et al, 2000), may be altering proliferative activity.

The highly proliferative MM in this series had other poor prognostic features including an increased frequency of monosomy 13 and/or deletion 13q chromosomal abnormalities and moderate to poorly differentiated tumour histology (Bartl grade II–III) (Tricot et al, 1995; Waldron et al, 1997). One of the surprising findings among the highly proliferative, cyclin D1-positive cases was the high incidence of women with IgA-secreting MM. Unfavourable cytogenetic karyotypes are known to correlate with the IgA isotype (Tricot et al, 1997). However, the specific risk for women in this group of tumours with cyclin D1 expression needs to be determined in a standardized clinical trial. In conclusion, the probable mechanism for cyclin D1 upregulation in MM with low or intermediate proliferative activity is through t(11;14). Cyclin D1 overexpression in highly proliferative tumours occurs independently of t(11;14). The data suggests that cyclin D1 upregulation does not appear to be directly linked to proliferation.


  1. Top of page
  2. Abstract
  3. Patients and methods
  4. Results
  5. Discussion
  6. References
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