Variations in the low levels of cyclin D1/BCL1 have prognostic value in chronic lymphocytic leukemia

Abstract

Cyclin D1 (CyD1)/BCL1 (PRAD1) is expressed at high levels in almost all cases of mantle cell leukemia/lymphoma (MCL) and in rare cases of chronic lymphocytic leukemia (CLL). The CyD1/BCL1 protein plays an important role in the progression of cells through the G1 phase of cell cycle. Most of the CyD1/BCL1 protein expression studies are performed using immunohistochemistry. We used a sensitive solid-phase radioimmunoassay (RIA) to quantify CyD1 protein expression in 199 patients with CLL. Of these 137 patients were previously untreated with the rest having had standard chemotherapeutic regimens including alkylating agents and fludarabine before being referred to our center. Median white cell count in these patients was 49×10³ /μl (range 3.0–438.5×10³ /μl), hemoglobin level 13.1 g/dl (range 5.2–17.3 g/dl), platelet count 157×10³ /μl (range 10–377×10³ /μl), age 58 (range 26–89), and β2-microglobulin 2.75 mg/dl (range 1.1–14.3). The median radioactivity (CPM) of mononuclear cells obtained from 56 normal individuals was assigned a value of 1. There was no significant variation in CyD1 levels among normal individuals (SD=0.12). While most CyD1 levels in MCL varied from 6.5 to 15.6, the median CyD1/BCL1 in CLL was 1.4 with 75th percentile under 2.12. Rare CLL cases (3.5%) showed levels between 4 and 8.83. When divided into two groups at the median level, patients with higher CyD1/BCL1 expression had shorter survival (P=0.03). This remained true when applied only to the previously untreated patients (P=0.05). Despite the relatively low expression, the CyD1/BCL1 levels in univariate analysis were as good or better predictors of survival than Binet (P=0.03) or Rai (P=0.05) staging. Furthermore, CyD1/BCL1 levels correlated with serum β2-microglobulin (P=0.001), white blood cell count (P=0.004) and hemoglobin levels at the time of collection (P=0.0003) but not with lymphocyte count, platelet count or age. The data demonstrate that CyD1/BCL1 is likely to play a significant role in the biology of CLL and can be used as a prognostic indicator. Further studies to clarify the role of CyD1 in the biology of CLL and its value as a prognostic indicator at the time of diagnosis are encouraged.

Introduction

Cyclin D1 (CyD1) is one of the proteins involved in cell cycle regulation in both normal and neoplastic cells [1], [2]. It is derived from the gene known variably as bcl-1, CCND1, or PRAD1, located on chromosome 11q13 [2]. The passage of cells from phases of the cell cycle is closely regulated by various factors influencing the transcription of cyclin genes, degradation of cyclin proteins, kinase activity and phosphorylation of various cell cycle proteins [1]. Cyclin D1, along with its corresponding cyclin D kinase (cdk), plays a major role in the transition of cells from the resting (G1) to the synthesis (S) phase of the cell cycle. This involves phosphorylation of the retinoblastoma tumor suppresser protein (pRB), which in turn releases transcription factors (including members of E2F proteins), initiating DNA replication [3]. The over-expression of the CyD1 protein, as a result of trans- or cis-activation of the gene, removes the normal regulatory constraints and permits uncontrolled proliferation and a transformation to a neoplastic phenotype [2]. No abnormality of the coding region of the CyD1 gene has been demonstrated, indicating that the normal gene product is the cause of the neoplastic change [2]. The t(11;14)(q13;q32) translocation, commonly associated with MCL, and occasionally with other B-lymphoproliferative disorders including CLL, hairy cell leukemia (HCL) and prolymphocytic leukemia (PLL) results in the translocation of the immunoglobulin heavy chain (IgH) inhancer into close proximity of the CyD1 gene leading to the over-expression of the gene and its protein product [4]. CyD1 over-expression has been shown in a number of human cancers including head and neck squamous cell carcinomas, esophageal carcinomas, lung and breast cancers. However, the most significant association of CyD1 over-expression is with MCL, where it has been shown to be present in the vast majority of cases [4], [5], [6], [7], [8].

Although the t(11;14)(q13;q32) translocation, and bcl-1 gene rearrangements have been reported in CLL, the presence of this translocation raises the question of misdiagnosis and the possibility of MCL [4], [9], [10], [11], [12], [13], [14]. Juliusson et al. reported chromosomal abnormalities involving 11q13 in 12 of 391(3%) CLL patients, ten of whom had t(11;14) [12]. Newman et al. noted the presence of bcl-1 gene rearrangement in five of 84 (6%) CLL patients who had atypical morphology and suggested the possibility of a different B-cell disease [11]. Yang et al. examined CyD1 protein expression in 15 MCLs and seven CLL/B-small lymphocytic lymphomas (SLL), using an immunohistochemical technique on formalin-fixed, paraffin-embedded tissue [14] and reported nuclear staining in 15 of 15 MCLs, and one of seven CLL/SLLs.

Using a highly sensitive and quantitative solid-phase radio-immunoassay (RIA) we quantified the CyD1 expression in 199 CLL patients, and compared this expression to 12 samples of MCL and normal mononuclear cells isolated from 56 normal individuals. We also correlated CyD1 expression in these CLL patients with various clinical features and outcome.