Althea McCourty

Immunohistochemical expression of cyclin D1, E2F-1, and Ki-67 in benign and malignant thyroid lesions.

Cyclin D1 and E2F‐1 proteins are essential for the regulation of the G1/S transition through the cell cycle. Cyclin D1, a product of the bcl‐1 gene, phosphorylates the retinoblastoma protein, releasing E2F‐1, which in turn activates genes involved in DNA synthesis. Expression patterns of E2F‐1 protein in thyroid proliferations have not been reported. This study used monoclonal antibodies for cyclin D1 and E2F‐1 proteins to immunostain sections of normal thyroid, hyperplastic (cellular) nodules, follicular adenomas, follicular carcinomas, and papillary carcinomas. The proliferation rate was examined using an antibody specific for the Ki‐67 antigen. Fluorescence in situ hybridization (FISH) methods and chromosome 11‐specific probes were also employed to determine chromosome copy number and to assess for evidence of amplification at the 11q13 locus in papillary and follicular carcinomas with cyclin D1 overexpression. Concurrent overexpression of Ki‐67, cyclin D1, and E2F‐1 was found in the majority of benign and malignant thyroid lesions, compared with normal thyroid tissue. Cyclin D1 up‐regulation was not due to extra copies of chromosome 11, or bcl‐1 gene amplification. Malignant tumours showed the highest expression for all three markers, particularly papillary carcinomas. E2F‐1 was detected at the same or slightly lower levels than cyclin D1. It was only found when cyclin D1 was overexpressed. Because cyclin D1 normally activates E2F‐1, up‐regulation of cyclin D1 may lead to E2F‐1 overexpression in benign and malignant thyroid lesions. Copyright

Cyclin D1 overexpression in Spitz nevi: an immunohistochemical study.

The morphologic distinction between Spitz nevus and malignant melanoma can be difficult. Because cyclin D1 has been reported to be overexpressed in malignant melanomas, but not in common acquired nevi, we hypothesized that cyclin D1 might be a useful marker to distinguish Spitz nevi from malignant melanoma. Thus, we assessed for cyclin D1 expression in 11 Spitz nevi (10 compound and 1 intradermal) and 9 malignant melanomas (4 Clark stages I-III and 5 Clark stages IV-V) using an immunohistochemical method and routinely fixed and processed tissues. The cyclin D1 results were arbitrarily divided into three groups: 0% to 10%, >10% to 25%, and >25%. We confirmed the observations reported previously by others that cyclin D1 is expressed in malignant melanomas but not in common acquired nevi. Unexpectedly, a relatively high number of cyclin D1-positive cells (i.e., >10%) was also found in all cases of Spitz nevus. However, unlike malignant melanoma, the cyclin D1 positivity in Spitz nevi was present in a zonal pattern. In other words, the number of cyclin D1-positive cells decreased as the lesion extended more deeply, with the number of positive cells in the reticular dermis being less than that in the papillary dermis. Fluorescence in situ hybridization methods were used to assess amplification of 11q13, the locus harboring the cyclin D1 gene, in four cases of Spitz nevus; all were disomic. Using the antibody MIB-1, we compared cyclin D1 expression to the proliferation rate in Spitz nevi. Despite the high cyclin D1 positivity, all Spitz nevi had a relatively low number of MIB-1-positive cells (mean=3.2%), which was significantly lower than that of malignant melanomas (mean=15.3%) (p < 0.001). Thus, unlike malignant melanoma, there appears to be a dissociation between cyclin D1 overexpression and cell proliferation in Spitz nevi.

Immunohistochemical Expression of Cell Cycle Proteins E2F-1, Cdk-2, Cyclin E, p27 kip1 , and Ki-67 in Normal Placenta and Gestational Trophoblastic Disease

The role of cell cycle protein expression in gestational trophoblastic disease is poorly understood. In this study we investigated the immunostaining patterns of G1 restriction point and G1-S regulatory proteins E2F-1, Cdk2, cyclin E, p27kip1, and the proliferation marker Ki-67 on routinely processed sections of 29 hydatidiform moles (10 partial moles and 19 complete moles, including 9 persistent moles), 7 choriocarcinomas, and 7 normal placentas. Ki-67 trophoblast staining decreased with increasing gestational age of the placenta, and showed maximal expression in gestational trophoblastic disease. Cyclin-dependent kinase activity, as reflected by Cdk2 expression patterns, also decreased with placental maturation. E2F-1 was uniquely expressed by trophoblasts of moles and choriocarcinoma. Cyclin E was maximally expressed by complete moles and choriocarcinomas, and showed an inverse relationship with the cyclin-dependent kinase inhibitor p27kip1. Abnormal trophoblastic proliferations may be mediated through interactions of Cdk-2, E2F-1, cyclin E, and p27kip1. Overexpression of cyclin E was associated with more aggressive forms of gestational trophoblastic disease. However, we did not find distinguishing features between complete moles that spontaneously resolved after evacuation and persistent moles that required chemotherapy. The different expression patterns of cyclin E and E2F-1 in partial and complete moles may be useful in distinguishing these two entities. Furthermore, loss of p27kip1 in malignant trophoblast may represent a necessary step in the development of choriocarcinoma.

Cyclin D1 and E2F‐1 immunoreactivity in bone marrow biopsy specimens of multiple myeloma: relationship to proliferative activity, cytogenetic abnormalities and DNA ploidy

Cyclin D1, encoded by the CCND1 gene, is immunohistochemically detectable in up to one‐third of cases of multiple myeloma (MM). To examine the mechanism of cyclin D1 overexpression, we compared cyclin D1 immunoreactivity with the results of conventional cytogenetics to determine if the t(11;14)(q13;q32) or other abnormalities of 11q11–14 explained cyclin D1 overexpression. Karyotypic abnormalities were found in 45 out of 67 (67%) MM cases; the t(11;14) was present in seven cases (10%). Additional 11q11–14 abnormalities were not identified. The t(11;14) correlated with cyclin D1 upregulation in low to intermediately proliferative MM, but was not present in highly proliferative tumours (assessed using bromodeoxyuridine labelling index). Cyclin D1 indirectly activates the transcription factor E2F‐1. In the bone marrow biopsy specimens of MM cases, E2F‐1 was concurrently expressed with cyclin D1 (P = 0·001), indicating that cyclin D1 is functional. However, as neither E2F‐1 nor cyclin D1 expression correlated with proliferative activity, the speculation that t(11;14) upregulates the CCND1 gene to induce higher proliferation and possibly more aggressive disease is not supported. We conclude that in low to intermediately proliferative MM cases, cyclin D1 is probably upregulated by t(11;14), but an alternative mechanism is more probable in highly proliferative MM.

Differential expression of cyclin D1 in breast papillary carcinomas and benign papillomas: An immunohistochemical study

OBJECTIVES Distinguishing intraductal papilloma from papillary carcinoma of the breast can be difficult using histologic criteria. Since cyclin D1, a G1 cell-cycle regulatory protein, is detectable immunohistochemically in a subset of breast carcinomas but not in benign breast tissues, we hypothesized that cyclin D1 immunoreactivity may be a marker for identifying papillary carcinoma. METHODS Using an immunohistochemical method, we assessed for cyclin D1 expression in 8 breast papillomas and 6 papillary carcinomas, all of which were formalin fixed, routinely processed, and paraffin embedded. Cyclin D1 positivity also was compared with the overall proliferation rate, which was assessed by using the proliferation marker Ki-67. In each case, a 200-cell count was performed to obtain the percentage of cells positive for these 2 markers. RESULTS The percentage of cyclin D1-positive cells was significantly higher in papillary carcinomas (89%+/-18%; range, 53%-98%) than in papillomas (8%+/-7%; range, 0%-19%). This difference was highly statistically significant (P < .0001). Although the difference in Ki-67 positivity between these 2 groups was also statistically significant (P = .01), separation of papillary carcinomas and papillomas by Ki-67 immunoreactivity was less clear because of overlapping values between groups: 13% +/-6%; range, 9% to 23% for papillary carcinomas versus 8%+/-2%; range, 6% to 12% for papillomas. CONCLUSIONS These results support the notion that cyclin D1 is a useful marker for distinguishing breast papillomas from papillary carcinomas. The marker Ki-67 is also helpful, but is less useful than cyclin D1, owing to the overlap in Ki-67 results in papillomas and papillary carcinomas.

DNA topoisomerase IIα in multiple myeloma: A marker of cell proliferation and not drug resistance

DNA topoisomerase IIα (topo IIα) is the target for a number of antineoplastic agents. Down-regulation of this enzyme is one form of drug resistance. Topo IIα is also involved in DNA replication and transcription and serves as an indicator of proliferation rate in many human malignancies. This study examines whether topo IIα is one of the mechanisms of chemoresistance commonly observed in multiple myeloma (MM) or alternatively, whether topo IIα is associated with tumor cell proliferation. Bone marrow (BM) biopsy sections from 72 cases of MM, stratified according to proliferative activity (bromodeoxyuridine uptake), were immunostained for topo IIα. Immunoreactivity with an addi- tional marker of drug resistance, glutathione-S-transferase π, and the proliferation marker Ki-67 were also examined. Topo IIα was expressed in 26 (36%) cases and correlated strongly with proliferative activity (P <.001). A role for drug resistance could not be supported, given this strong relationship with proliferation and the finding that glutathione-S-transferase π expression in 57 (78%) cases was independent of topo IIα immunoreactivity. Topo IIα was identified in 91 to 100% of highly proliferative tumors, as evaluated by bromodeoxyuridine uptake or Ki-67 reactivity, respectively. Proliferation also correlated with the histologic grade of the MM. Therefore, topo IIα immunoreactivity is primarily a marker of cell proliferation in MM and as such is likely to have prognostic significance. Highly proliferative tumors are most likely to be sensitive to chemotherapeutic protocols using anti–topo IIα agents.