Cyclin D1 by flow cytometry as a useful tool in the diagnosis of B-cell malignancies
Introduction
The chronic B-cell malignancies comprise a group of disorders, which despite being related to each other, have different therapeutic approaches and outcomes [1], [2]. The diagnosis is based on clinical features, morphological examination of blood or bone marrow films, lymph node or bone marrow histology and immunophenotyping of the malignant cells [1], [3]. In some instances, uncertainty of diagnosis becomes an issue, which may be resolved by cytogenetic and molecular analysis. The t(11;14)(q13;q32) is the common chromosomal abnormality in MCL, occurring in over 90% of cases. This translocation, also found in some cases of B-PLL, MM and occasional SLVL [4], [5], joins the IGH locus on 14q32 to the BCL-1 locus on chromosome 11q13 [6], [7], [8].
MCL has features in common with both low grade and intermediate grade B-cell lymphomas [9], [10]. In lymph node sections MCL has either a vague nodular or a diffuse growth pattern. Cytologically the tumours are composed of uniformly small or medium sized lymphoid cells [11], [12] which express CD5 as well as most B antigens, but lack CD23 and CD10 and are sIgM+, and often IgD+ [12], [13].
Cyclin D1 is a nuclear protein, being one of the D family of cyclins [2], [14]. Expression of the different members of this family is tissue and cell-type specific; whereas cyclin D1 is expressed mainly by epithelial cells and some mesenchymal cells, cyclins D2 and D3 are expressed by myelomonocytic and lymphoid cells [15]. The cyclin D1 gene, designated CCND1, encodes a small 295 amino acid protein [5], [7]. Overexpression of cyclin D1 can be induced either by amplification of the chromosomal region of the cyclin D1 gene at 11q13 as described in some solid tumours [16], or by the chromosomal translocation (11;14)(q13;q32). As a consequence overexpression of cyclin D1 mRNA as well as protein can be detected in most haematological tumours carrying this translocation [15].
Cycin D1 being a G1 cyclin, is a major positive regulator of the cell cycle progression at the G1-S phase transition and associates with other proteins such as the cyclin-dependant kinase 4 (CDK-4) with which it forms a complex [17], [18]. Overexpression of the cyclin D1 results in an abnormal proliferation of the cells with shortened G1 phase and less dependence on growth factors [19], [20], [21], [22].
Cyclin D1 expression is used as a diagnostic marker for MCL and for differentiating, along with other parameters, between MCL and the other B-cell disorders. A variety of reliable diagnostic methods were developed over the past few years for the detection of t(11;14) bearing malignancies and cyclin D1 overexpression. These methods include: standard cytogenetics, Southern blotting and hybridisation [5], Northern blotting [2], FISH [23], [24], immunoblotting [15], RT-PCR [25] and immunohistochemistry [9]. The first three methods involve techniques, which are often time consuming. We describe here a simple dual parameter flow cytometric procedure to detect cyclin D1 at the protein level, which gives comparable results to the RT-competitive PCR method.
Section snippets
Samples and controls
Peripheral blood and bone marrow heparinised samples were taken from 93 patients with various B-cell disorders with blood involvement. The cases included were: 13 MCL, 13 SLVL, 30 CLL, 15 low grade B-NHL (cases in which no further specification of diagnosis was available), 5 FL, 4 B-PLL, 4 LPL, 7 HCL, and 2 MM. The diagnosis was based on morphology and cytology according to the French-American-British (FAB) proposals, on immunophenotyping and on histology when available. Diagnosis of MCL was
Expression of D1, D2 and D3 cyclins in cell lines
By flow cytometry, the Granta519 and Karpas620 cell lines exhibited cyclin D1 expression with a high MFI which is the difference between the fluorescence intensity of the Ab stained cells and of the isotypic control, a higher value being obtained with the G519 cell line (Fig. 1(b) and Table 2). They also displayed a strong intensity of D1 expression by RT-PCR with relatively less pronounced cyclins D2 and D3 in the G519 cell line (Fig. 2(a)). The pattern of cyclin D1 expression seen by flow
Discussion
We analysed the protein levels of cyclin D1 by flow cytometry using the MoAb 5D4, and the mRNA levels of cyclins D1, D2 and D3 by RT-PCR in a group of B-cell malignancies and positive control cell lines.
Despite the high peaks of cyclin D1 positive cells observed in the cell line G519 and some MCL (Fig. 1), the overall intensity of fluorescence as observed on performing the same experiments but without propidium iodide and analysing on histogram plots (log scale) was usually of weak-moderate
Acknowledgements
This work was supported by Trust funds from the Royal Marsden NHS Trust and the Leukaemia Research Fund. We are grateful to Dr. Martin Dyer for providing cell lines. We thank Drs. V. Brito-Babapulle and A. Gruszka-Westwood for their help with the FISH analysis. Our gratitude also goes to Benjamin Hilditch for his assistance in the final preparations of the manuscript. M.O. Elnenaei provided the concept, design, assembled the data and analyzed it and drafted the paper. D.M. Jadavel provided
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