Mantle cell lymphomas lack expression of p27Kip1, a cyclin-dependent kinase inhibitor

Abstract

p27Kip1 is a cyclin-dependent kinase inhibitor that regulates the decision to enter S phase or withdraw from the cell cycle. In resting cells, the level of p27Kip1 provides an inhibitory threshold above which G1 cyclin D/E/cyclin-dependent kinases accumulate before activation; however, in cycling cells, p27Kip1 protein is sequestered by high levels of active cyclin D/cyclin-dependent kinase 4 complexes. As a group, the cyclin-dependent kinase inhibitors have been proposed to act as tumor suppressor genes, and several members have been implicated in the pathogenesis of a variety of human cancers. We examined p27Kip1 expression in 116 non-Hodgkin's lymphomas including 50 cases of MCL (40 typical and 10 blastic variants), 21 follicular lymphomas, 20 diffuse large B-cell lymphomas, 16 chronic lymphocytic leukemias, 8 marginal zone B-cell lymphomas, and 1 splenic marginal zone lymphoma, and correlated its expression with that of the proliferation marker Ki67 (MiB1) and with p53. p27Kip1 gene structure was analyzed by Southern blot in the group of MCLs. In all cases of non-Hodgkin's lymphoma other than MCL, p27Kip1 expression was inversely related to the proliferation index as measured by Ki67. In contrast, in typical MCL, p27Kip1 expression was negative in 35 of 40 (88%) cases, irrespective of the proliferative rate (median 15%; range 2 to 90%). Paradoxically, in the blastic variant of MCL, 8 of 10 (80%) cases showed expression of p27Kip1, despite a high proliferation rate (median 60%; range 32 to 100%). However, the staining in most of the cases was less intense than in the reactive T lymphocytes. Deletions of p27Kip1 gene were not found in any of the 25 cases examined. p53 expression was found in 15 of 50 cases of MCL: 7 of 10 (70%) in the blastic variant and 8 of 40 (20%) in the typical MCL (70% vs. 20%, P < 0.0045). These results demonstrate that MCLs, in contrast to other non-Hodgkin's lymphomas and normal lymphoid tissue, fail to correlate p27Kip1 expression with the proliferation rate. This peculiar uncoupling of p27Kip1 protein expression from the proliferation rate may be related to the high levels of cyclin D1 expressed in MCL and is likely to have profound effects on cell cycle regulation and contribute to the pathogenesis of MCL.

Figure 1.

p27^Kip1 protein expression in normal lymphoid tissue (tonsil). Germinal centers that contain a high percentage of proliferating cells show only scattered cells expressing p27^Kip1, whereas most cells in the mantle zone and in the interfollicular T-cell area express high levels of p27^Kip1. Immunoperoxidase; ×100.

Figure 2.

A: p27^Kip1 protein expression in a case of CLL. The neoplastic cells show a diffuse strong nuclear positivity. Immunoperoxidase; ×400. B: p27^Kip1 protein expression in a case of DLBCL. The large neoplastic cells are completely negative for p27^Kip1, whereas the small reactive T lymphocytes are positive. Immunoperoxidase; ×400.

Figure 3.

p27^Kip1 and cyclin D1 expression in a case of typical MCL. A: MCL cells staining positively for cyclin D1. The residual germinal center shows a negative reaction. Immunoperoxidase; ×200. B: In contrast to cyclin D1, the negative p27^Kip1 staining of the MCL cells accentuates the mantle zone (perifollicular) growth pattern. Note the negative staining in the reactive germinal center and the positive staining in the residual T-cell area. Immunoperoxidase; ×100). C: p27^Kip1 in a typical case of MCL with a diffuse growth pattern. The tumor cells are negative, whereas the intermingled T lymphocytes are strongly positive. Immunoperoxidase; ×400. D: Double staining for p27^Kip1 (red) and CD3 (black) in a case of MCL reveals that all of the p27^Kip1-positive cells within the tumor co-express CD3. The lymphoma cells are negative for both p27^Kip1 and CD3. Immunoperoxidase; ABC-alkaline phosphatase method (red) and ABC-3,3′-diaminobenzidine method (black); ×400; inset, ×1000. E Strong p27^Kip expression in the majority of tumor cells in a case of a large-cell variant of MCL. Immunoperoxidase; ×400. F, G, and H: p27^Kip, Ki67, and p53 expression in a case of blastic variant of MCL. F: p27^Kip1 is weakly expressed by the majority of tumor cells despite the high proliferation rate. Note the stronger positivity of the intermingled T lymphocytes. Immunoperoxidase; ×400. G: Ki67 is expressed by the vast majority of the tumor cells. Immunoperoxidase; ×400. H: p53-positive staining in tumor cells. Immunoperoxidase; ×400.

Figure 4.

Southern blot analysis of 16 cases of typical MCL (Lanes 1 to 16). BamHI-digested DNA separated by agarose gel electrophoresis and blotted into a nylon membrane was hybridized with a ³²P-labeled p27^Kipprobe. All cases show a germline configuration without evidence of rearrangement or deletion of p27^Kip1gene. The varying signal intensities are due to unequal loading of DNA as confirmed by hybridization with a control probe (data not shown). Lane 17 corresponds to placental DNA.