Determination of cyclin D1 and CD20 mRNA levels by real-time quantitative RT-PCR from archival tissue sections of mantle cell lymphoma and other non-Hodgkin's lymphomas

Abstract

Cyclin D1 overexpression is a valuable marker for the diagnosis of mantle cell lymphoma (MCL). We used a real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) method to quantify levels of cyclin D1, CD20, and cyclophilin A mRNA in manually microdissected, paraffin-embedded tissue sections using an ABI 7700 qRT-PCR system. The study group included 21 cases of MCL and 37 cases of other types of B-cell non-Hodgkin's lymphoma. Cyclin D1 mRNA copy number was normalized to CD20 and cyclophilin A mRNA and evaluated statistically by analysis of variance. The relative cyclin D1 levels were similar whether normalized to CD20 or cyclophilin A, indicating that CD20 levels are stable and can be used as a B-cell-specific normalizer. Statistically significant differences were found in the median levels of cyclin D1 mRNA (expressed as % CD20 mRNA) among cases of MCL (87.6), small lymphocytic lymphoma (9.9), follicular lymphoma (2.4), diffuse large B-cell lymphoma (5.9), marginal zone B-cell lymphoma (39.8), and Burkitt lymphoma (7.1) (P < 0.05). We conclude that qRT-PCR can be used to quantify cyclin D1 mRNA levels in archival tissue sections. Normalization of cyclin D1 to a B-cell-specific marker more accurately reflects overexpression by MCL than other methods that normalize using constitutively expressed mRNA species.

Figure 1.

Scatter plot showing CD20 mRNA copy number as a function of cyclophilin A copy number (log-scale). The correlation coefficient (R) and P value were calculated using the logarithms of the absolute copy numbers.

Figure 2.

Relative cyclin D1 mRNA levels expressed as percentage of CyPA (A) or CD20 (B) mRNA copy number. The end points of the solid bars represent 25th and 75th percentile. Median is indicated by the star symbol. The inserted bar graphs show the median relative cyclin D1 mRNA levels for each normalizer (stippled bars).

Figure 3.

CD20 mRNA expression levels in various NHL (median of CD20/CyPA mRNA copy numbers).