Detection of cyclin D1 (bcl-1, PRAD1) overexpression by a simple competitive reverse transcription-polymerase chain reaction assay in t(11;14)(q13;q32)-bearing B-cell malignancies and/or mantle cell lymphoma.

In mantle cell lymphoma, the t(11;14)(q13;q32) and its molecular counterpart, bcl-1 rearrangement, are consistent features and lead to cyclin D1 (bcl-1, PRAD1) proto-oncogene overexpression. In order to detect cyclin D1 overexpression, we developed a simple assay involving a reverse transcription followed by competitive polymerase chain reaction (PCR). A single upstream primer was derived from a homologous region between cyclin D1 and the other D-type cyclins, cyclins D2 and D3, while three downstream primers were specific to their respective D-type cyclins. Because the upstream primer was shared in PCR amplification of the three sequences, each PCR product served as a competitor and the quantification of the target was made by comparison of the intensity of the three products. With this assay we analyzed 45 hematopoietic cell lines and 40 clinical specimens. Cyclin D1 was rarely expressed in lymphoid cell lines except in t(11;14)(q13;q32)-bearing B-cell malignancies and/or mantle cell lymphoma, which expressed cyclin D1 predominantly. In myeloid cell lines, the levels of cyclin D1 expression varied and never exceeded the sum of cyclin D2 and D3 levels. Cyclin D3 was ubiquitously expressed while cyclins D1 and D2 were differentially used. The observations suggest that human cyclin D3 may play a fundamental role in hematopoiesis and that cyclins D1 and D2 may have different lineage- or differentiation-dependent functions. With this assay, small aliquots of clinical specimens such as 100 microL peripheral blood were enough to detect cyclin D1 overexpression without a well-controlled standard. The technique was validated as highly comparable with Northern analysis. This rapid and reliable detection of cyclin D1 overexpression may have practical clinical utility in the analysis and management of B-cell malignancies.