Value of Interphase FISH for the Diagnosis of t(11;14)(q13;q32) on Skin Lesions of Mantle Cell Lymphoma

Pierre Dubus, MD, PhD, Paul Young, MD, Marie Beylot-Barry, MD, PhD, Marc A. Belaud-Rotureau, PhD, Philippe Courville, MD, Béatrice Vergier, MD, PhD, Marie Parrens, MD, Bernard Lenormand, MD, PhD, Pascal Joly, MD, PhD, Jean P. Merlio, MD, PhD

Disclosures

Am J Clin Pathol. 2002;118(6) 

In This Article

Results

The epidermis was normal in all cases. In case 1, a nodular infiltrate was seen throughout the dermis with small nodules of lymphoid cells without germinal centers in the papillary and reticular dermis. Lymphoid cells were clustered around vessels and adnexa (Figure 1C). A bottom-heavy arrangement was seen as the deep dermis contained larger nodules with occasional germinal centers outlined by immunostaining of dendritic cells by CD23 (Figure 1D). In case 2, the lesions were restricted to the upper dermis with small perivascular aggregates (Figure 1E). In case 3, a dense and diffuse infiltration of the deep dermis and subcutis was seen [Figure 1F]). In cases 1 and 3, the infiltrate was monotonous with small to medium-sized centrocytic cells with scant cytoplasm, irregular cleaved nuclei, and coarse chromatin (Figure 1F). In case 2, a high proliferative index, as determined by MIB-1 expression, was associated with pleomorphic blastoid morphologic features of lymphoid cells with immature chromatin (Figure 1E, inset).

Lymphoid cells expressed CD20 and CD43 antigens and were negative for CD3, CD10, and CD23. The CD5 antigen was detected on centrocytic cells in cases 1 and 2 with lower intensity than on reactive T cells (Figure 1G) but remained negative on several sections of skin specimens from case 3. In cases 1 and 3, a parallel lymph node biopsy also revealed MCL involvement with identical cytologic and phenotypic features.

Nuclear immunoreactivity for cyclin D1 was detected in the lymphoma cells of case 3 in skin and lymph node specimens (Figure 1H). In case 1, faint cyclin D1 immunoreactivity was detected in MCL lymph node cells but not on skin sections. In the latter, cells of the basal layers of the epidermis also remained negative. This suggested degradation of the protein, so the study was interpreted as not contributive ( Table 2 ). In case 2, epithelial cells were positive, but the MCL infiltrate was not immunoreactive for cyclin D1 (data not shown).

IgH
Gene and Detection of t(11;14) Breakpoint by Genomic PCR

As summarized in Table 3 , a monoclonal rearrangement of the IgH gene was detected in all specimens (skin, blood, bone marrow, and lymph node) for case 1, in skin and blood specimens for case 2, and in skin and lymph node specimens for case 3. For each case, the monoclonal FR3-JH dominant band was identical between samples by its migration on gel electrophoresis (data not shown). In case 3, a t(11;14) breakpoint was amplified by genomic PCR in skin and lymph node specimens but not in blood specimens, in accordance with the absence of a monoclonal IgH fragment in this sample (Figure 2). In cases 1 and 2, no t(11;14) breakpoint was amplified by genomic PCR in any tested sample ( Table 3 ).

Detection of t(11;14) genomic breakpoint by polymerase chain reaction (PCR) amplification with MCL1-JH primers. Upper panel, ethidium bromide stain after electrophoresis of PCR products. Lower panel, hybridization with the MCL2 probe after Southern blot. 0, water as a negative control; T, 2 positive controls; 1 and 2, skin specimens from cases 1 and 2; and from case 3, 3a, blood; 3b, lymph node; and 3c, skin. MCL, mantle cell lymphoma.

As shown in Figure 3, RT-PCR revealed overexpression of cyclin D1 transcripts in comparison with cyclin D2 and D3 transcripts in PBLs and lymph node specimens in case 1. Expression of cyclin D1 also was detected in skin samples in cases 1, 2, and 3 but was not significantly higher than those of cyclin D2 and D3. Moreover, the parallel study of 6 control skin specimens from 6 patients with nonspecific inflammatory dermatoses also showed constitutive expression of cyclin D1, D2, and D3 genes (Figure 3). Therefore, RT-PCR study of skin specimens was considered not appli-cable ( Table 2 ).

Competitive reverse transcriptase-polymerase chain reaction study of cyclin D1, D2, and D3 transcripts. Gel electrophoresis of polymerase chain reaction products. The size and position of the different transcripts are indicated by arrows. Note the overexpression of cyclin D1 transcripts in blood (1b) and lymph node (1n) specimens from case 1 compared with healthy donor lymphocytes (N). In skin specimens from cases 1 (1s), 2, and 3, no difference is observed between the levels of the cyclin transcripts, as also seen in skin specimens of nonspecific inflammatory dermatoses (4-9). 0, water as template; MW, 100-base-pair ladder.

For each section, 300 well-hybridized, separately located, interphase nuclei were evaluated. Two main categories were identified: normal nuclei with 2 red and 2 green signals (Figure 1I) and abnormal nuclei considered t(11;14)+ when 1 green and 1 red signal were fused (overlapping signals) or colocalized (touching signals) (Figure 1J). The cutoff value or false-positive threshold was estimated at 5%, corresponding to the mean plus 3 SD of 5 independent counts of 300 nuclei on sections of reactive lymphadenitis in which random distribution of chromosomes 11 and 14 can generate overlapping or touching signals in the absence of t(11;14). In case 1, nuclei from touch preparations of frozen lymph node and skin showed 73% and 30% abnormal nuclei, respectively, while formalin-fixed sections of lymph node showed 65% abnormal nuclei. In cases 2 and 3, sections from formalin-fixed, paraffin-embedded skin biopsy specimens were available and showed 50% and 78% abnormal nuclei, respectively.

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