In Situ Hybridization and Reverse Transcription–Polymerase Chain Reaction for Cyclin D1 mRNA in the Diagnosis of Mantle Cell Lymphoma in Paraffin-Embedded Tissues

Abstract

Mantle cell lymphoma (MCL) is characterized by the chromosomal translocation t(11;14), which involves rearrangement of the bcl-1 proto-oncogene to the immunoglobulin heavy chain gene and results in overexpression of cyclin D1 mRNA. In this study, we evaluated the diagnostic relevance of three methods that may be helpful in the diagnosis of MCL: in situ hybridization (ISH) and a stringent reverse transcriptase-polymerase chain reaction (RT-PCR) protocol for cyclin D1 mRNA, and immunohistochemistry for cyclin D1 protein. The study group included 37 paraffin-embedded specimens (25 from lymph nodes and 12 from extranodal tissues) from 30 patients. MCL diagnosis was performed according to the Revised European-American Classification of Lymphoid Neoplasms. Twenty-nine patients with non-MCL lymphoproliferative disorders comprised the control group. Biotin-labeled ISH was performed in 28 cases of MCL, 24 (86%) of which were found to be positive. As shown by ISH in extranodal tissues, cyclin D1 mRNA was present not only in neoplastic lymphoid cells, but in other cell types as well. For this reason, RT-PCR results were considered reliable for MCL diagnosis only on informative material (from tissues that do not normally express cyclin D1); this method was evaluated as positive in 16 of 18 (89%) MCL cases. Cyclin D1 immunopositivity was present in 20 of 29 (69%) MCL cases. No members of the control group were found to express cyclin D1 mRNA by either ISH or RT-PCR under the stringent conditions used. In conclusion, stringent RT-PCR for cyclin D1 expression can be helpful in MCL diagnosis in paraffin-embedded material from lymph nodes. ISH is a sensitive method for cyclin D1 mRNA detection; its sensitivity is superior to that of cyclin D1 immunohistochemistry and similar to that of the stringent RT-PCR used. ISH is very specific as well, clearly more specific than RT-PCR, because it allows the correlation of molecular findings with morphology. This method can be applied on all types of paraffin-embedded tissues and provides an accurate tool for MCL diagnosis.

Real-Time RT-PCR Assay for Quantifying Cyclin D1 mRNA in B-Cell Non-Hodgkin's Lymphomas

“…Others have used RT-PCR methods to assess for cyclin D1 in MCL with results in general agreement with our own (18,23,(25)(26)(27)(28). In some of these studies, the presence or absence of cyclin D1 was considered the endpoint without quantification (23).…”

“…Previous studies using Northern blot analysis, immunohistochemical methods, and semiquantitative RT-PCR methods have suggested that nonneoplastic lymphoid tissues are negative for cyclin D1 (4,18,23,24). However, it is clear from the data presented in this study that cyclin D1 mRNA is expressed at low levels in reactive lymphoid tissues when assessed using the real-time RT-PCR methods, as also has been described by others (25).…”

“…Reactive GI biopsy specimens also had appreciable cyclin D1 levels (median cyclin D1/GAPDH ratio 24.2), similar to that of reactive LN specimens. The most likely explanation for the cyclin D1 levels detected in the GI specimens is the presence of epithelial cells (as well as endothelial cells) that are known to express cyclin D1 (19,23), as glandular cells were plentiful in the routinely processed histologic sections of these specimens. In contrast, uninvolved PB and BM specimens have extremely low levels of cyclin D1 expression, presumably related to the relatively low degree of vascularity and absence of epithelial cells in these specimens.…”

“…In some of these studies, the presence or absence of cyclin D1 was considered the endpoint without quantification (23). In other studies, traditional semiquantitative methods RT-PCR methods have been employed (18, 26 -28).…”

A Comparative Analysis of FISH, RT-PCR, PCR, and Immunohistochemistry for the Diagnosis of Mantle Cell Lymphomas

“…It has been individualized both in the REAL and in the World Health Organization classifications because it represents a distinct clinical pathological entity with a special aggressive course among small B-cell lymphomas (2,3). It frequently involves lymph node, blood, bone marrow, and spleen (4,5). The neoplastic cells typically exhibit a centrocytic morphology and coexpress pan-B-cell markers CD5 and CD43 but lack CD23 and CD10.…”