The P16-Cyclin D1/CDK4-Prb Pathway and Clinical Outcome in Epithelial Ovarian Cancer1

4152 Vol. 5, 4152–4157, December 1999 Clinical Cancer Research

The p16-Cyclin D1/CDK4-pRb Pathway and Clinical Outcome in Epithelial Ovarian Cancer1

Tomoaki Kusume, Hiroshi Tsuda,2 INTRODUCTION Masami Kawabata, Takeshi Inoue, Ovarian cancer is the most common cancer in women to be Naohiko Umesaki, Tomoko Suzuki, and diagnosed at an advanced stage and is the leading cause of death from gynecological malignancy. Fifty % of patients, after opti- Kumio Yamamoto mal surgical debulking and a pathologically complete response Departments of Obstetrics and Gynecology [T. K., H. T., M. K., to primary chemotherapy, will develop a recurrence of the tumor K. Y.] and Pathology [T. I.], Osaka City General Hospital, Osaka 534-0021; Department of Obstetrics and Gynecology, Osaka City and will die within 2 years. The development of additional University Medical School, Osaka [N. U.]; and Department of prognostic factors, closely related to tumor cell biology, is Biostatistics and Epidemiology, Faculty of Medicine, University of essential for identification of patients with a particularly poor Tokyo, Tokyo [T. S.], Japan prognosis. Recent genetic and biochemical investigations of the

molecular mechanisms governing the G1 to S progression in mammalian cells have demonstrated an important role for D- ABSTRACT type cyclins and their partner kinases CDK43 and CDK6 (1–4). A significant positive association has been reported be- When activated by cyclin D1, CDK4 is able to phosphorylate tween p16 expression and clinical outcome for epithelial pRb, leading to the release of associated proteins like E2F that ovarian cancer patients. However, there is a reciprocal cor- have the capability to activate genes necessary for cell progres- relation between genetic alterations of single members of the sion through the G1 phase (4). p16 controls cell cycle prolifer- p16-cyclin D1/CDK4-pRb pathway (G1 pathway). Simulta- ation during G1 by inhibiting the ability of cyclin D/CDK4 and neous evaluation of these four elements may produce a cyclin D/CDK6 complexes to phosphorylate pRb (5). The com- better prognostic factor than p16 alone. We studied the ponents of the p16-cyclin D/CDK-pRb pathway (G1 pathway) prognostic significance of the G1 pathway in 59 epithelial are frequently found to be altered in various types of cancers ovarian cancer patients undergoing surgery and platinum- (6–11). In ovarian cancer, the loss of p16 expression was based chemotherapy by immunohistochemical technique. reported to be detected in 11–37% of studied cases (12–15). It Abnormal expression of p16 or pRb was defined by negative has been reported that overexpression of p16 might be an nuclei staining, and that of CDK4 and cyclin D1 was defined important early event in ovarian cancer (16) and that high by 50% nuclear staining. An abnormal G1 pathway was expression of p16 is associated with poor prognosis in ovarian indicated in cases that have at least one abnormality among cancer patients (13, 14). However, findings of loss of p16 these four elements. Abnormal expression of p16, pRb, and expression are generally thought to be abnormal. Heyman et al. cyclin D1/CDK4 was observed in 33.9, 3.4, and 15.3% of (17) reported a statistically significant correlation between in- studied cases, respectively. Abnormal G1 pathway was de- activation of p15/p16 by homozygous deletion or intragenic tected in 49.2% (29 of 59) of all cases. The patients with mutation and poor prognosis. These findings seem contradic- normal G1 pathway tended to achieve a higher complete tory. response rate (81.0%) to chemotherapy, compared with pa- In solid tumors, there is a reciprocal correlation between tients with abnormal G1 pathway (55.0%); however, there genetic alterations of single members of the p16-cyclin ؍ was no significant difference (P 0.1001) between the two D/CDK4-pRb pathway (18–20). It has been reported that hy- groups. Univariate analyses identified advanced stage [haz- pophosphorylated active pRb can repress p16 expression, ؍ ards ratio (HR), 3.665; P 0.0218], histological low grade whereas inactivation of pRb by phosphorylation leads to p16 ؍ (HR, 3.625; P 0.0066), and abnormal G1 pathway (HR, expression (21). Fang et al. (22) reported that expression of p16 ؍ 2.935; P 0.03) as prognostic factors for overall survival. induced transcriptional down-regulation of the Rb gene. Con- The G1 pathway might help as a prognostic factor to select sistent with these findings, CDKN2 gene deletion and Rb defi- high-risk patients. ciency are reported to be inversely correlated in many types of tumors (8, 18). In addition, a strong association between altered cyclin D1 and pRb expression has been reported in esophageal tumors (23). Muller et al. (24) have demonstrated that the cell cycle-dependent expression of cyclin D1 in tumor cell lines Received 4/15/99; revised 9/23/99; accepted 9/27/99. The costs of publication of this article were defrayed in part by the requires the presence of a functional pRb. Masciullo et al. (25) payment of page charges. This article must therefore be hereby marked reported a direct relationship between the expression levels of advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported by grants from Osaka City General Hospital. 2 To whom requests for reprints should be addressed, at Department of Obstetrics and Gynecology, Osaka City General Hospital, 2-13-22 3 The abbreviations used are: CDK, cyclin-dependent kinase; Rb, reti- Miyakojimahondori, Miyakojima, Osaka 534-0021, Japan. Phone: 06- noblastoma; pRb, Rb protein; IHC, immunohistochemistry; CR, com- 6929-1221; Fax: 06-6929-2041. plete response; OS, overall survival.

Table 1 Association of abnormal expression of p16, CDK4, cyclin D1, and pRb and clinicopathological features Clinicopathological features n p16 pRb CDK4 Cyclin D1 Pathway Clinical stage 1/2 25 9 (36.0%) 2 (8.0%) 4 (16.0%) 2 (8.0%) 14 (56.0%) 3/4 34 11 (32.3%) 0 (0%) 5 (14.7%) 1 (2.9%) 15 (44.1%) Histological type Serous 25 10 (40.0%) 1 (4.0%) 3 (12.0%) 1 (4.0%) 13 (52.0%) Mucinous 6 3 (50.0%) 0 (0%) 0 (0%) 0 (0%) 3 (50.0%) Endometrioid 15 3 (20.0%) 0 (0%) 4 (26.7%) 2 (13.3%) 7 (46.7%) Clear cell 11 3 (27.3%) 1 (9.1%) 2 (18.2%) 0 (0%) 5 (45.5%) Unclassified 2 1 (50.0%) 0 (0%) 0 (0%) 0 (0%) 1 (50.0%) Histological grade

G1 18 7 (38.9%) 0 (0%) 0 (0%) 1 (5.6%) 7 (38.9%) G2 26 9 (34.6%) 2 (7.7%) 5 (19.2%) 0 (0%) 14 (53.8%) G3 15 4 (26.7%) 0 (0%) 4 (26.7%) 2 (13.3%) 8 (53.3%) Total 59 20 (33.9%) 2 (3.4%) 9 (15.3%) 3 (5.1%) 29 (49.2%)

cyclin D1 and CDK4. The expression levels and activities of were reacted with one of the following primary antibodies these proteins can modulate each other. We believe it important at 4°C overnight: (a) rabbit anti-p16 polyclonal antibody to evaluate these four elements simultaneously. Additionally, to (PharMingen, San Diego, CA); (b) mouse anti-pRb monoclonal our knowledge, there are no reports in which p16, cyclin D1, antibody (PharMingen, San Diego, CA); (c) rabbit anti-CDK4 CDK4, and pRb were examined simultaneously in epithelial polyclonal antibody (Santa Cruz Biotechnology, Inc., Santa ovarian cancer. In this study, we investigated the prognostic Cruz, CA); (d) mouse anti-cyclin D1 monoclonal antibody importance of all these key components of this G1 checkpoint by (Medical and Biological Laboratories Co., Nagoya, Japan), or IHC approaches in epithelial ovarian cancer. nonimmunized rabbit serum. After rinsing, the sections were incubated for 30 min with mouse or rabbit EnVisionϩ Peroxi- MATERIALS AND METHODS dase (Dako, CA). The peroxidase activity for p16, pRb, CDK4, Clinical Samples. All of the specimens analyzed in this and cyclin D1 was visualized by applying diaminobenzidine study were obtained from patients who were newly diagnosed chromogen containing 0.05% hydrogen peroxide for 2–10 min from 1994 to 1998 at Osaka City General Hospital, Osaka, at room temperature. The sections were then counterstained with Japan. Paraffin-embedded tissue of 59 ovarian cancers (stage 1, hematoxylin. Positive and negative control experiments were 19; stage 2, 6; stage 3, 29; and stage 4, 5) were collected. Of the performed for each tumor staining. 59 tumors, histology types included 25 serous, 6 mucinous, 11 Interpretation of IHC Staining. The slides were read by clear cell, 15 endometrioid, and 2 undifferentiated. All surgical two professional pathologists who were blinded to the clinical staging and debulking procedures were performed by experi- outcome of the patients. Tumors were scored as p16-negative if enced gynecologists using standard techniques. Histological di- all malignant cells had no nuclear staining and surrounding agnosis was confirmed by microscopic examination of the normal stromal cells showed adequate nuclear staining as a H&E-stained sections according to WHO criteria. Clinical positive internal control. Tumors were regarded as p16-positive stages were determined according to the International Federa- tion of Gynecology and Obstetrics system. All patients with if any malignant cells had nuclear staining. Small lymphocytes, stages 1c, 2, 3, and 4 cancer received postoperative chemother- which showed no nuclear staining of p16, were used as a apy with a platinum-based regimen. Postoperative chemother- negative internal control (26). The same criteria were used for apy from 1994 to 1997 was comprised of nine cycles of CAP the evaluation of pRb staining. Negative nuclear staining was (cyclophosphamide-Adriamycin-platinum) therapy composed considered as abnormal expression of p16 and pRb. Evaluation of 500 mg/m2 cyclophosphamide, 50 mg/m2 doxorubicin, and of CDK4 and cyclin D1 positives was performed by semiquan- Ϫ 50 mg/m2 cisplatin. The regimen from 1998 was TP (Taxol- titative analyses. The following scale was used (27): ,no Ͻ platinum) therapy composed of 180 mg/m2 paclitaxel over 3 h, immunoreactive tumor cells detectable or 5% of the tumor followed by 75 mg/m2 cisplatin or AUC5 carboplatin. CR was cells were positive with a weak intensity; 1ϩ, 5–50% of the clinically defined by normalization of the 35 units/ml CA125, tumor cells were positive with a strong intensity; and 2ϩ, Ͼ50% resolution of computed tomographic scan abnormalities, and a of the tumor cells were positive with a strong intensity. Strong normal physical examination after completion of first-line nuclear staining(2ϩ) was considered as abnormal expression of chemotherapy. CDK4 and cyclin D1. Abnormal G1 pathway was indicated in IHC of p16, pRb, CDK4, and Cyclin D1 Proteins. His- cases that have at least one abnormality among these four tological sections were affixed to glass slides, dewaxed, and elements. rehydrated. Autoclave unmasking process (10 min at 121°C in Statistical Analysis. Univariate differences between cat- 10 mM citrate buffer, pH 6.0) was used. The sections were then egoric variables were evaluated by using Fisher’s exact test. OS incubated in 3% hydrogen peroxide for 10 min at room temper- distribution was calculated by using the Kaplan-Meier method, ature to quench endogenous peroxidase activity. The sections and univariate Cox regression analyses were used to identify

Fig. 1 Typical images of p16, pRb, CDK4, and cyclin D1 staining. A, positive p16 immunostaining. B, negative p16 immunostaining. The section shows negative nuclear staining of the tumor cells. C, positive pRb immunostaining. D, negative pRb immunostaining. Note that stromal cells show distinct nuclear staining (arrowheads) of p16 and pRb, which provide positive internal controls for both proteins. E, strong positive CDK4 immunostaining. F, strong positive cyclin D1 immunostaining. a–f, ϫ200.

variables associated with OS. All Ps are for two-sided signifi- type, or histological grade. Twenty-six of 29 patients (89.7%) cance tests. with abnormal pathway had only one abnormal factor. There tended to be a reciprocal correlation between genetic alterations RESULTS of single members of the p16-cyclin D1/CDK4-pRb pathway. p16, pRb, CDK4, and Cyclin D1 Expression in Primary Nine of 39 (23.1%) patients with p16 expression had abnormal Tumor Samples. Associations of abnormal expression of expression of pRb, cyclin D1 or CDK4, and four of these nine p16, CDK4, cyclin D1, and pRb and clinicopathological features cases (abnormal pathway with p16 expression) died within 3 are shown in Table 1. Loss of p16 and pRb and strong expres- years. If p16 expression is divided into two groups (strong sion of CDK4 and cyclin D1 were 33.9, 3.4, 15.3, and 5.1%, expression, Ͼ50% of the tumor cells were positive; weak ex- Ͻ respectively. Abnormal G1 pathway was detected in 49.2% (29 pression, 50% of the tumor cells were positive), four of nine of 59) of all cases. Abnormal expression of these four factors cases (44.4%) with abnormal expression of cyclin D1/CDK4 was not significantly associated with clinical stage, histological had strong p16 expression, and only one of 30 cases (3.3%) with

Fig. 2 a, overall Kaplan-Meier survival curves of epithelial ovarian cancer patients show significant difference in survival between normal and ϭ ϭ abnormal G1 pathway (P 0.0213). b, the p16 status was not associated with survival (P 0.1245). CNSR, censored.

Table 2 Statistical analysis of the effect of various prognostic associated with clinical stage (P ϭ 0.4348), histological type factors on OS (P ϭ 0.7948), histological grade (P ϭ 0.7710), and residual Univariate Hazards disease status (P ϭ 0.7710). Typical images of p16, pRb, Variablea P ratiob 95% CIc CDK4, and cyclin D1 staining are shown in Fig. 1.

Age 0.0124 1.057 1.012–1.104 Relationship between the Status of the G1 Pathway and Stage 0.0218 3.655 1.208–11.056 the Clinical Responses to Platinum-based Chemotherapy as Histology 0.4469 1.437 0.565–3.650 First-Line Chemotherapy. Forty-one of 59 patients were as- Grade 0.0148 3.070 1.245–7.570 p16 0.1373 1.984 0.804–4.902 sessable for CR on the basis of persistent elevation of the CDK4 0.0990 2.561 0.838–7.813 CA125 level or measurable disease on computed tomographic cyclinD1 0.1015 3.436 0.784–15.15 scan postoperatively. There were no significant differences in pRb 0.8833 1.164 0.154–8.850 mean age, clinical stage, histological type, histological grade, G1 pathway 0.0300 2.935 1.110–7.762 and residual disease status between the abnormal G pathway a м Ͻ 1 Age, 60 versus 60 years; Stage, III/IV versus I/II; Histology, and normal G pathway groups. The CR rate to chemotherapy in nonserous versus serous; Grade, III versus I/II; p16, negative versus 1 positive; CDK4, м50% versus Ͻ50% cells positive; cyclin D1, м50% the cases with assessable disease postoperatively was 68.3% (28 Ͻ versus 50% cells positive; pRb, negative versus positive; G1 pathway, of 41 patients with assessable disease postoperatively). Eighty- abnormal versus normal. one % of patients with normal G pathway achieved CR, com- b Ͻ 1 Hazard ratio refers to risk of death, with values 1.0 indicating pared with 55.0% of patients with abnormal G pathway. The reduced risk. 1 c CI, confidence interval. patients with normal G1 pathway tended to achieve higher CR rate, compared with the patients with abnormal G1 pathway; however, the difference between the two groups was not significant (P ϭ 0.1001). normal pathway had strong p16 expression. All patients were Relationship between the Status of the G1 Pathway and divided into two groups, abnormal G1 pathway and normal G1 OS Rate. For the entire cohort, the OS was 67.8% (median pathway. There were no significant differences in mean age follow-up, 22 months). As shown in Fig. 2a, OS in loss of p16 (54.4 Ϯ 11.9 versus 54.4 Ϯ 14.7; t test, P ϭ 0.9892). Fisher’s was 55.0% (median follow-up, 20 months) and OS in expression exact tests indicated that the status of the G1 pathway was not of p16 was 74.4% (median follow-up, 27 months), which does

not constitute a significant difference (P ϭ 0.1245). The status predictor of OS, despite the short follow-up period. In addition,

of pRb, CDK4, or cyclin D1 was not also associated with OS all four patients with stage 1 or 2 who died had abnormal G1 (data not shown). However, OS of 80.0% (median follow-up, pathways. However, p16, CDK4, cyclin D1, or pRb status alone

27.0 months) for patients in the normal G1 pathway group was was not associated with clinical outcome. The G1 pathway was significantly superior to the OS of 55.1% (median follow-up, 19 found to be a better prognostic predictor of ovarian cancer than ϭ months) for patients in the abnormal G1 pathway group (P p16, CDK4, cyclin D1, or pRb alone. High expression of p16

0.0213; Fig. 2b). By univariate analysis, abnormal G1 pathway, was reported to be associated with poor prognosis in ovarian advanced stage, and histological grade were significant predic- cancer patients (13, 14). These findings seem to contradict our tors of poor OS (Table 2). results. We have the following speculations. In this study, the abnormalities of p16 and cyclin D1/CDK4 tended to be reciprocal. Nine of 39 (23.1%) patients with p16 expression had DISCUSSION abnormal expression of pRb, cyclin D1 or CDK4, and four of

It has been reported that loss of expression of p16 in human these nine cases (abnormal G1 pathway with p16 expression) ovarian tumors was 11–37% (12–15). Some reports demon- died within 3 years. In our study, four of nine cases (44.4%) strated that loss of p16 expression was detected mainly in with abnormal expression of cyclin D1/CDK4 had strong p16

mucinous and endometrioid types and advanced stage cancers expression; however, only 1 of 30 cases (3.3%) with normal G1 (13–15, 28). In this study, loss of p16 expression was 33.9%, pathway had strong p16 expression. Strong p16 expression and there was no significant relationship between p16 status and tended to be associated with abnormal expression of cyclin clinical stage, histological grade, or histology. We did not ex- D1/CDK4. Yao et al. (38) reported that CDK4 overexpression amine the CDKN2 gene status; however, CDKN2 gene muta- accompanied elevated p16 status and that elevated p16 levels tions are thought to be rare events in ovarian cancer (12, 14, might be the result of compensatory up-regulation of this protein 28–30). Concerning pRb, most ovarian epithelial tumors (86– to counteract CDK4 overexpression in primary soft-tissue sar- 96%) show high pRb expression with IHC (31–33). In our coma. It may be that some of the cases with high p16 expression series, loss of pRb was detected only in 3.3% (3 of 59). There in former reports (13, 14) also had abnormal expression of pRb,

are few reports about the abnormalities of CDK4 and cyclin D1 cyclin D1, or CDK4. Status of the G1 pathway was associated in ovarian cancer (10, 25). In these reports, overexpression of with clinical outcome in ovarian cancer patients. However, p16, CDK4 and cyclinD1 were 14% (7 of 48) and 18% (12 of 65), CDK4, cyclin D1, or pRb status was not a good clinical predic- respectively, by Northern blot analysis, and these did not appear tor because some patients with expression of p16 also exhibited to be associated with clinical outcome. In our study, strong abnormal expression of pRb, cyclin D1, or CDK4. In conclu-

expressions of CDK4 and cyclin D1 were 15.3 and 5.1%, sion, the G1 pathway is a useful prognostic predictor in ovarian respectively, by IHC, and in the strong expression group, 40% cancer. (4 of 10) died within 3 years because of tumor progression. Most cases (89.7%: 26 of 29) with abnormal G1 pathway had only one ACKNOWLEDGMENTS abnormal factor of p16, CDK4, cyclin D1, or pRb. The abnor- We are grateful to Dr Kazuhiko Nakagawa of the Kinki University malities of p16 and cyclin D1/CDK4 tended to be reciprocal. and Kathrine Allikian of Enzyme Bio-Systems, Ltd. for their critical To our knowledge, there have been no reports in which the review of the manuscript and to Fumi Kaibara, Keiko Higa, and Nozomi correlation between the response to chemotherapy and G1 path- Tsuji for technical assistance. way was examined in ovarian cancer. In our studies, patients with normal G pathway tended to achieve a higher CR rate with 1 REFERENCES a platinum-based regimen, compared with patients with abnor- 1. Kamb, A. Cell-cycle regulators and cancer. Trends Genet., 9: 136– mal G1 pathway. Effects of alterations in cell cycle genes on 140, 1995. cytotoxicity of chemotherapeutic agents remain unclear. It was 2. Sherr, C. J., and Roberts, J. M. Inhibitors of mammalian G1 cyclin- reported that p16-mediated G1 arrest prevented platinum- dependent kinases. Genes Dev., 9: 1149–1163, 1995. induced cell death (34, 35). Hochhauser et al. (36) demonstrated 3. Strauss, M., Lukas, J., and Bartek, J. Unrestricted cell cycling and that there was an increased fraction of cells in the S and G2 cancer. Nat. Med., 1: 1245–1246, 1995. phases of the cell cycle among cells expressing higher levels of 4. Weinberg, R. A. The retinoblastoma protein and cell cycle control. cyclin D1. Cytotoxicity assays revealed a statistically significant Cell, 81: 323–330, 1995. increase in resistance to methotrexate in cells expressing high 5. Serrano, M., Hannon, G. J., and Beach, D. A new regulatory motif in levels of cyclin D1; however, there was no difference in resist- cell-cycle control causing specific inhibition of cyclin D/CDK4. Nature (Lond.), 366: 704–707, 1993. ance to doxorubicin and paclitaxel. Fukuoka et al. (37) reported 6. Cairns, P., Polascik, T. J., Eby, Y., Tokino, K., Califano, J., Merlo, that p16INK4 expression was closely associated with the in- A., Mao, L., Herath, J., Jenkins, R., and Westraw, W. Frequency of creased sensitivity of an ectopic p16INK4-expressing non-small homozygous deletion at p16/CDKN2 in primary human tumors. Nat. cell lung cancer cell line with p16 homozygous deletion to Genet., 11: 210–212, 1995. topoisomerase I inhibitors. Effects of alterations in cell cycle 7. Nakagawa, K., Conrad, N. K., Williams, J. P., Johnson, B. E., and genes on cytotoxicity may depend on the type of drug. We Kelley, M. J. Mechanism of inactivation of CDKN2 and MTS2 in non-small cell lung cancer and association with advanced stage. Onco- performed platinum-based combination chemotherapy including gene, 11: 1843–1851, 1995. cyclophosphamides and doxorubicin or paclitaxel. Further ex- 8. Kinoshita, I., Dosaka-Akita, H. T., Mishina, T., Akie, K., Nishi, M., amination will be required to resolve this problem. Hiroumi, H., Hommura, F., and Kawakami, Y. Altered p16INK4 and In this study, G1 pathway status proved to be a significant retinoblastoma protein status in non-small cell lung cancer: potential

synergistic effect with altered p53 protein on proliferative activity. 25. Masciullo, V., Scambi, G., Marone, M., Giannitellic, C., Fer- Cancer Res., 56: 5557–5562, 1996. randina, G., Benedetti Panici, P., and Mancuso, S. Altered expression of 9. Shinozaki, H., Ozawa, S., Ando, N., Tsuruta, H., Terada, M., Ueda, cyclin D1 and CDK4 genes in ovarian carcinomas. Int. J. Cancer, 74: M., and Kitajima, M. Cyclin D1 amplification as a new predictive 390–395, 1997. classification for squamous cell carcinoma of the esophagus, adding 26. Kratzke, R. A, Greaters, T. M., Rubins, J. B., Maddaus, M. A., gene information. Clin. Cancer Res., 2: 1155–1161, 1996. Niewoehner, D. E., Niehans, G. A., and Geradts, J. Rb and p16INK4A 10. Barbieri, F., Cagnoli, M., Ragni, N., Pedulla, F., Foglia, G., and expression in resected non-small cell lung tumors. Cancer Res., 56: Alama, A. Expression of cyclinD1 correlates with malignancy in human 3415–3420, 1996. ovarian tumours. Br. J. Cancer, 75: 1263–1268, 1997. 27. Michalides, R., van Veelen, N., Hart, A., Loftus, B., Wientjens, E., 11. Zhang, T., Nanney, L. B., Luongo, C., Lamps, L., Heppner, K. J., and Balm, A. Overexpression of cyclin D1 correlates with recurrence in DuBois, R. N., and Beauchamp, R. D. Concurrent overexpression of a group of forty-seven operable squamous cell carcinomas of the head cyclin D1 and cyclin dependent kinase 4 (CDK4) in intestinal adenomas and neck. Cancer Res., 55: 975–978, 1995. from multiple intestinal neoplasia (Min) mice and human familial ade- nomatous polyposis patients. Cancer Res., 57: 169–175, 1997. 28. Ichikawa, Y., Yoshida, S., Koyama, Y., Hirai, M., Ishikawa, T., Nishida, M., Tsunoda, H., Kubo, T., Miwa, M., and Uchida, K. Inacti- 12. Marchini, S., Codegoni, A. M., Bonazzi, C., Chiari, S., and Broggini, M. Absence of deletions but frequent loss of expression of vation of p16/CDKN2 and p15/MTS2 genes in different histological p16INK4 in human ovarian tumours. Br. J. Cancer, 76: 146–149, 1997. types and clinical stages of primary ovarian tumors. Int. J. Cancer, 69: 466–470, 1996. 13. Dong, Y., Walsh, M. D., and McGuckin, M. A. Increased expression of cyclin-dependent kinase inhibitor 2 (CDKN2A) gene product 29. Schuyer, M., Staveren, I. L., Klijn, J. G., vd Burg, M. E., Stoter, G., p16INK4A in ovarian cancer is associated with progression and un- Henzen-Logmans, S. C., Foekens, J. A., and Berns, E. M. Sporadic favourable prognosis. Int. J. Cancer, 74: 57–63, 1997. CDKN2 (MTS1/p16ink4) gene alterations in human ovarian tumors. 14. Fujita, M., Enomoto, T., Haba, T., Nakashima, R., Sasaki, M., Br. J. Cancer, 74: 1069–1073, 1996. Yoshino, K., Wada, H., Buzard, G. S., Matsuzaki, N., Wakasa, K., and 30. Shih, Y. C., Kerr, J., Liu, J., Hurst, T., Khoo, S. K., Ward, B., Murata, Y. Alteration of p16 and p15 genes in common epithelial Wainwright, B., and Chenevix-Trench, G. Rare mutations and no hy- ovarian tumors. Int. J. Cancer, 74: 148–155, 1997. permethylation at the CDKN2A locus in epithelial ovarian cancer. Int. J. 15. Langosch, K. M., Ocon, E., and Becker, G. p16/MTS1 inactivation Cancer, 70: 508–511, 1997. in ovarian carcinomas: high frequency of reduced protein expression 31. Dodson, M. K., Cliby, W. A., Xu, H. J., Delacey, K. A., Hu, S. X., associated with hyper-methylation or mutation in endometrioid and Keeney, G. L., Li, J., Podratz, K. C., Jenkins, R. B., and Benedict, W. F. mucinous tumors. Int. J. Cancer, 79: 61–65, 1998. Evidence of functional RB protein in epithelial ovarian carcinomas 16. Shigemasa, K., Hu, C., West, C. M., Clarke, J., Parham, G. P., despite loss of heterozygosity at the RB locus. Cancer Res., 54: 610– Parmley, T. H., Korourian, S., Baker, V. V., and O’Brien, J. J. p16 613, 1994. overexpression: a potential early indicator of transformation in ovarian 32. Kim, T. M., Benedict, W. F., Xu, H. J., Hu, S. X, Gosewehr, J., carcinoma. J. Soc. Gynecol. Investig., 4: 95–102, 1997. Velicescuo, M., Yin, E., Zheng, J., D’Ablaing, G., and Dubeau, L. Loss 17. Heyman, M., Rasool, O., Borgonovo Brandter, L., Liu, Y., Grander, of heterozygosity on chromosome 13 is common only in the biologically D., Soderhall, S., Gustavsson, G., and Einhorn, S. Prognostic impor- more aggressive subtypes of ovarian epithelial tumor and is associated tance of p15INK4B and p16INK4 gene inactivation in childhood acute with normal retinoblastoma gene expression. Cancer Res., 54: 605–609, lymphocytic leukemia. J. Clin. Oncol., 1512–1520, 1996. 1994. 18. Bartkova, J., Lukas, J., Guldberg, P., Alsner, J., Kirkin, A. F., 33. Dong, Y., Walsh, M. D., and McGuckin, M. A. Reduced expression Zeuthen, J., and Bartek, J. The p16-cyclin D/Cdk4-pRb pathway as a functional unit frequently altered in melanoma pathogenesis. Cancer of retinoblastoma gene product (pRB) and high expression of p53 are Res., 56: 5475–5483, 1996. associated with poor prognosis in ovarian cancer. Int. J. Cancer, 74: 407–415, 1997. 19. He, J., Allen, J. R., Collins, V. P., Allalunis-Turner, M. J., Godbout, R., Day, R. S., III, and James, C. D. CDK4 amplification is an alterna- 34. Hama, S., Heike, Y., Naruse, I., Takahashi, M., Yoshioka, H., Arita, tive mechanism to p16 homozygous deletion in glioma cell lines. Cancer K., Kurisu, K., Goldman, C. K., Curiel, D. T., and Saijo, N. Adenovirus- Res., 54: 5804–5807, 1994. mediated p16 gene transfer prevents drug-induced cell death through G1 arrest in human glioma cells. Int. J. Cancer, 77: 47–54, 1998. 20. Schauer, I. E., Siriwardanaa, S., Langan, T. A., and Sclafani, R. A. Cyclin D1 overexpression vs. retinoblastoma inactivation: implications 35. Stone, S., Dayananth, P., and Kamb, A. Reversible, p16-mediated for growth control evasion in non-small and small cell lung cancer. Proc. cell cycle arrest as protection from chemotherapy. Cancer Res., 56: Natl. Acad. Sci. USA, 91: 7827–7831, 1994. 3199–3202, 1996. 21. Li, Y., Nichols, M. A., Shay, J. W., and Xiong, Y. Transcriptional 36. Hochhauser, D., Schnieders, B., Ercikan-Abali, E., Gorlick, R., repression of the D-type cyclin-dependent kinase inhibitor p16 by the Muise-Helmericks, R., Li, W. W., Fan, J., Banerjee, D., and Bertino, retinoblastoma susceptibility gene product pRB. Cancer Res., 54: 6078– J. R. Effect of cyclin D1 overexpression on drug sensitivity in 6082, 1994. human fibrosarcoma cell lines. J. Natl. Cancer Inst., 88: 1269–1275, 22. Fang, X., Jin, X., Xu, H. J., Liu, L., Peng, H. Q., Hogg, D., Roth, 1996. J. A., Yu, Y., Xu, F., Bast, R. C., Jr., and Mills, G. B. Expression of p16 37. Fukuoka, K., Adachi, J., Nishio, K., Arioka, H., Kurokawa, H., induces transcriptional downregulation of the RB gene. Oncogene, 16: Fukumoto, H., Ishida, T., Namoto, T., Yokote, H., Tomonari, A., 1–8, 1998. Narita, N., Yokota, J., and Saijo, N. p16INK4 expression is associ- 23. Jiang, W., Zhang, Y. J., Kahn, S. M., Hollstein, M. C., Santella, ated with the increased sensitivity of human non-small cell lung R. M., Lu, S. H., Harris, C. C., Montesano, R., and Weinstein, I. B. cancer cells to DNA topoisomerase I inhibitors. Jpn. J. Cancer Res., Altered expression of the cyclin D1 and retinoblastoma genes in human 88: 1009–1016, 1997. esophageal cancer. Proc. Natl. Acad. Sci. USA, 90: 9026–9030, 1993. 38. Yao, J., Pollock, R. E., Lang, A., Tan, M., Pisters, P. W., Goodrich, 24. Muller, H., Lukas, J., Schneider, A., Warthoe, P., Bartek, J., Eilers, D., EI-Naggar, A., and Yu, D. Infrequent mutation of the p16/MTS1 M., and Strauss M. Cyclin D1 expression is regulated by the retinoblas- gene and overexpression of cyclin-dependent kinase 4 in human primary toma protein. Proc. Natl. Acad. Sci. USA, 91: 2945–2949, 1994. soft-tissue sarcoma. Clin. Cancer Res., 4: 1065–1070, 1998.

Downloaded from clincancerres.aacrjournals.org on September 24, 2021. © 1999 American Association for Cancer Research. The p16-Cyclin D1/CDK4-pRb Pathway and Clinical Outcome in Epithelial Ovarian Cancer

Tomoaki Kusume, Hiroshi Tsuda, Masami Kawabata, et al.

Clin Cancer Res 1999;5:4152-4157.

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