D-cyclins are cell cycle regulatory proteins whose expression and distribution in human tissue types and neoplasms are incompletely characterized. In the current work, we show that among the three D-cyclin proteins, cyclin D1 has the most restricted pattern of expression, whereas cyclin D2 exhibits more widespread expression in a variety of hematopoietic and non-hematopoietic tissues. The expression pattern of cyclins D2 and D3 is in sharp contrast to the known tissue distribution of cyclin D1, which is not found in normal hematopoietic cells. Our findings are in keeping with studies in mouse splenic B-cells that showed cyclins D2 and D3 but not D1 as crucial for the proliferative capacity of mitogen-stimulation of B cells.19
In normal hematopoietic tissues, cyclins D2 and D3 were expressed in a proportion of B- and T- cell subsets as ascertained by double-labeling experiments. Although cyclin D3 was found in fewer lymphoid cells than cyclin D2, the pattern of expression in different lymphoid cell compartments in normal hematopoietic organs (tonsil, lymph node, thymus and spleen) was similar to each other. Most importantly, a significant proportion of germinal center B-cells lacked cyclin D2 and D3 staining. Although cyclin D2 is essential for B-cell receptor-mediated signaling and proliferation of naive B-cells20,21
, its low abundance in germinal center B-cells is supported by prior gene expression profiling studies.22
The mechanism of how some germinal center B-cells proliferate in the absence of these key cell cycle regulators of the G1/S transition that controls cell proliferation, is as yet unknown.
The expression of cyclin D2 and D3 proteins in normal hematopoietic tissues was mirrored by their expression across the hematopoietic neoplasms studied, with some exceptions. Cyclin D2 was the most widely expressed D-cyclin and was found in almost all subtypes of lymphomas including a subset of classical Hodgkin lymphoma. Although normal myeloid precursors and blasts in the bone marrow did not show significant amount of staining for cyclin D2, all acute myeloid leukemias and subsets of B- and T- lymphoblastic lymphoma/leukemias showed staining for cyclin D2 in a subset of blasts. Cyclin D3 staining was detected in erythroid precursors and in megakaryocytes in the normal bone marrow, and its overexpression was also detected in a subset of the blasts in all acute myeloid leukemias and in B- and T- lymphoblastic lymphoma/leukemias. Given that cyclin D2 and D3 expression is minimal or absent in normal marrow precursors, these findings raise the possibility that their expression may be activated during leukemogenesis and are overexpressed in acute leukemias. Since a significant proportion of acute leukemias showed overexpression of both cyclins D2 and D3, these results suggest the possibility of coordinate regulation of cyclin D2 and D3 in these leukemias.
Translocation t(11;14) (q13;q32) at the cyclin D1 locus results in the overexpression of the cyclin D1 protein which accelerates lymphoma cells through the G1/S transition of the cell cycle leading to dysregulated proliferation.5,6
In cyclin D1-negative mantle cell lymphomas, there is overexpression of cyclin D2 or D3 proteins.7
Furthermore, knockdown of cyclin D1 mRNA in mantle cell lymphoma cell lines have shown a modest compensatory increase in the expression of cyclin D2 mRNA and protein, suggesting the possibility for coordinate regulation of the expression of D-cyclins.23
This finding is in keeping with gene targeting experiments in mice deficient in D-cyclins that suggested the presence of functional redundancy among mammalian D-cyclins.24
Several recent reports have described translocations targeting the CCND2
loci in cyclin D1-negative mantle cell lymphomas: t(12;14)(p13;q32)/IGH-CCND2
We performed FISH analysis to further interrogate our cohort of mantle cell lymphoma cases: 16 of 18 cases harbored the CCND1/IGH
translocation and all expressed the cyclin D1 protein. The single case in our series that showed weak cyclin D1 but strong cyclin D2 staining was found to harbor the CCND1/IGH
translocation. Therefore, we did not pursue additional cytogenetic analyses as our cases would be unlikely to yield a known or novel translocation involving CCND2
However, it would be of interest to characterize the cytogenetic abnormalities of additional cyclin D1-negative mantle cell lymphomas to better understand the spectrum of chromosomal defects acquired by this rare type of lymphoma.
Apart from mantle cell lymphoma, cyclin D1 expression was detected in a four cases (2%) of diffuse large B-cell lymphoma. These cases showed intense nuclear staining in the vast majority of lymphoma cells. In two cases where material was available for cytogenetic/FISH studies, neither the CCND1/IGH
translocation nor abnormalities associated with the CCND1
locus was detected. Complete karyotypic analysis was not performed due to the lack of fresh tissue. Rare cases of cyclin D1-positive diffuse large B-cell lymphoma have been previously reported.25,26
In a series of 231 cases of diffuse large B-cell lymphoma, Ehinger and colleagues described 10 cases (4%) that showed staining for cyclin D1; the majority of these cases showed aberrancies involving the CCND1
locus and one case showed the CCND1/IGH
In a single case reported by Rodrigues-Justo and colleagues, aberrancies involving the CCND1
locus but not the translocation were detected.26
Our findings are in keeping with these prior observations that cyclin D1 is expressed in rare cases of diffuse large B-cell lymphoma. These findings underscore the importance of using an integrated approach to diagnosis that involves morphologic assessment, clinical input and a panel of immunostains (including BCL2, BCL6, IgM, and IgD in addition to cyclin D1) to distinguish among large cell or blastoid variants of mantle cell and diffuse large B-cell or other high grade B-cell lymphomas.
Our results in a variety of lymphoma subtypes show that immunostaining for cyclin D2 and D3 lacks specificity with regard to a particular subtype of lymphoma; staining for cyclin D2 and D3 was found in a subset of almost all types of B- and T- cell lymphomas including lymphoblastic and classical Hodgkin lymphomas. As important cell cycle regulatory molecules, the overexpression of cyclin D2 and D3 proteins in lymphomas and leukemias with a propensity for increased proliferation is not unexpected. This is reflected in our data on lymphoblastic lymphoma and T and NK-cell lymphoma subtypes where all cases that showed a high degree of proliferation (>50% by Ki-67 staining) also showed high levels of expression of cyclin D2 and/or D3. Of particular interest is that a significant proportion of low grade B-cell lymphomas also express cyclins D2 and D3. In these cases, the expression of D-cyclins did not correlate with increased proliferation. This finding is particularly relevant to grade 1 follicular lymphomas with increased proliferation, a group of lymphomas reported to follow a more aggressive clinical course.27
The four cases in our cohort that belong to this category showed no expression of D-cyclin proteins. These findings may be suggestive of additional as yet unidentified cyclins that may be involved in the regulation of proliferation in lymphoma subtypes or other mechanisms of cell cycle regulation at play. Therefore, it should be emphasized that the presence of cyclin D2 or D3 staining in a low grade B cell lymphoma should not be interpreted as compatible with a diagnosis of cyclin D1-negative mantle cell lymphoma. This latter entity is a rare diagnosis and as such, morphologic features, other immunohistologic markers (CD5, IgM, IgD), and FISH studies for abnormalities involving the CCND1
locus should be correlated before the diagnosis of this rare entity is rendered. In addition, since cyclin D2 and D3 proteins are expressed in lymphoid and myeloid leukemias, caution should be exercised in utilizing these stains for bone marrow diagnosis. Potential pitfalls include cases in which the marrow is replaced by a diffuse proliferation of intermediate cell size where a concurrent aspirate smear is not available for sufficient morphologic assessment; this proliferation could potentially lead to confusion of a lymphoblastic or myeloid leukemia with mantle cell lymphoma. Therefore, given the expression of cyclins D2 and D3 in a variety of lymphoid subtypes, it is necessary for careful assessment of these markers in the context of other findings.
The overexpression of cyclin D2 and D3 proteins in a variety of subsets of lymphomas raises the possibility that dysregulation of specific biological pathways associated with the cell cycle may play a role in their pathogenesis and prognosis. For example, we found that among ALK-positive anaplastic large cell lymphomas, 60% overexpressed cyclin D2 and 25% overexpressed cyclin D3. This result corresponds with previous gene expression profiling studies that identified the expression of cyclin D3 in ALK-positive but not in ALK-negative ALCL.28
These findings further suggest that ALK-positive anaplastic large cell lymphoma may exploit a different mechanism of promoting G1/S transition than its ALK-negative counterpart. Given the significant difference in clinical outcome between these entities, the overexpression of cyclin D3 may prove to be a marker of superior outcome in this disease. However, further work on sufficiently well-characterized ALK-positive and ALK-negative anaplastic large cell lymphoma cases is needed to formally prove this hypothesis.
We previously identified CCND2
as the best predictor of inferior survival in diffuse large B-cell lymphoma patients in a multivariate model based on the expression of six-genes as measured by quantitative RT-PCR.11,12
Immunohistochemical staining for cyclin D2 was also found to be associated with an inferior outcome in patients with diffuse large B-cell lymphoma treated with anthracycline-containing chemotherapy.13
In a set of 143 cases of diffuse large B-cell lymphoma that we have previously evaluated for markers of germinal center and non-germinal center derivation,15,16,18,29
hierarchical cluster analysis showed that cyclins D2 and D3 staining exhibit protein expression profiles that are similar to each other across the diffuse large B-cell lymphoma cases. This pattern of expression of cyclins D2 and D3 in diffuse large B-cell lymphoma suggests that the relationship of these proteins to prognostic stratification may be associated with cell cycle control rather than the cell of origin. However, in our cohort of diffuse large B-cell lymphoma cases the degree of proliferation as measured by Ki-67 staining and the expression of D-cyclins did not yield a significant correlation. In addition, cases that did not express any of the three D-cyclins showed a range of proliferation from 10 to 70%. Therefore, the proliferative capacity of diffuse large B-cell lymphomas is likely governed by complex and as yet unknown mechanisms that may not be restricted to cell cycle control. Of note, prior gene expression profiling studies showed low abundance of cyclin D2 in germinal center B-cells,22
although our data shows variable expression of cyclin D2 protein in germinal center B-cells. Our findings also differ from a previous report that found that cyclin D3 but not cyclin D2 was expressed in germinal center B-cells.30
Whether the differential expression of cyclins D2 and D3 proteins is associated with patient outcome warrants further investigation in a cohort of diffuse large B-cell lymphoma patients uniformly treated with Rituximab-based immunochemotherapy as these regimens have been shown to be of improved benefit in patients with diffuse large B-cell lymphoma.31–34