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

Abstract

Mantle cell lymphoma (MCL) diagnosis first relies on morphology and phenotype that may overlap with other B-cell lymphomas. Therefore, the demonstration of t(11;14)(q13;q32), the cytogenetic hallmark of MCL, is considered of diagnostic value. By studying a series of 35 MCL with characteristic morphology and phenotype (CD5؉, CD10؊, CD20؉, CD23؊), we have evaluated the applicability and the sensitivity of interphase fluorescence in situ hybridization (FISH) for t(11;14) detection and other techniques: (1) polymerase chain reaction (PCR) for amplification of t(11;14) genomic breakpoint, (2) competitive RT-PCR for the detection of cyclin D1 transcripts overexpression, and (3) immunohistochemistry (IHC) for cyclin D1 protein detection. Tissues from different origins were analyzed: lymph nodes (n ‫؍‬ 24), spleen (n ‫؍‬ 3), digestive biopsy (n ‫؍‬ 3), tonsils (n ‫؍‬ 3), and skin (n ‫؍‬ 2). Interphase FISH was performed either on touch preparations (n ‫؍‬ 11) and frozen (n ‫؍‬ 9) or paraffin sections (n ‫؍‬ 15). FISH analysis detected t(11;14) in 34/35 cases (97%) and demonstrated a recurrent CCND1 amplification in t(11;14)؉ nuclei of the three blastoid MCL variants of our series. Genomic PCR analysis, hampered by the scattering of 11q13 breakpoints, was positive in only 13/35 cases (37%). RT-PCR analysis was applicable on nonepithelial tissues (27/35) and showed cyclin D1 transcript overexpression in all tested cases (27/35). IHC for cyclin D1 protein was performed either on frozen (n ‫؍‬ 12) or on paraffin sections (n ‫؍‬ 23), and its sensitivity was higher on paraffin sections (91%) than on frozen sections (25%). A cyclin D1 protein immunoreactivity was observed in 24/35 cases (69%). Our study emphasizes on the use of FISH analysis for the direct detection of t(11;14) because its applicability and sensitivity largely exceeded those of other techniques. It may also provide some informations on secondary cytogenetic changes of potential clinical relevance.

Fluorescence in situ hybridization: Method of choice for a definitive diagnosis of mantle cell lymphoma†

“…Utilizing the IGH/CCND1 probes, Li et al identified positive results in 51 MCL cases studied but not in any of the 12 control cases [9]. Belaud-Rotureau et al demonstrated CCND1 by FISH in 34 of 35 MCL cases [12]. Our study confirms the results of the previous studies in terms of sensitivity and specificity of FISH for the diagnosis of MCL.…”

“…Cytogenetics, Southern blotting, and PCR have been used for this purpose. The sensitivity of cytogenetics and Southern blotting ranges from 37% to 80% [7,9,12]. The PCR has an even lower sensitivity in the range of 50-60% because of the large number of 11q13 breakpoints scattering over a region of more than 120 kb [26,27].…”

“…However, conventional cytogenetics and polymerase chain reaction are of relatively low sensitivity [7][8][9], while the detection of RNA transcripts by reverse transcription PCR and by in situ hybridization is of low specificity [7]. Thus far, the fluorescence in situ hybridization technique (FISH) seems to be most promising in terms of sensitivity and specificity [9][10][11][12]. The goal of the current study is to compare FISH with other common modalities that can be routinely used in clinical laboratories.…”

Fine-needle cytology and flow cytometry immunophenotyping and subclassification of non-hodgkin lymphoma

“…Dual-color FISH analysis was performed using an IGH-CCND1 probe set (Vysis Inc., Downer's Grove, IL), as described elsewhere. 20,21 FC FNC specimens were processed within 2 hours, and the cell suspensions were washed twice by centrifugation for 5 minutes at 2500 revolutions per minute, removing the supernatant fluid and adding 400 L of PBS. The final suspension was divided into four or more tubes when sufficient cells were available.…”

“…6). 19,21 The phenotype CD3ϩ, CD56ϩ, CD19Ϫ, CD10Ϫ/CD23Ϫ was specific for natural killer cell lymphoma. 22,25 In large cell and anaplastic NHL, the phenotype CD19ϩ, CD19/CD5Ϫ, CD19/CD10Ϫ, CD19/CD23Ϫ, CD38/ CD56Ϫ was observed the most frequently.…”