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Clinical Cancer Research Vol. 11, 3265-3273, May 1, 2005
© 2005 American Association for Cancer Research

Imaging, Diagnosis, Prognosis

Expression of Cell Cycle–Regulated Proteins pRB2/p130, p107, E2F4, p27, and pCNA in Salivary Gland Tumors: Prognostic and Diagnostic Implications

Giuseppe Russo1,3, Alessandra Zamparelli1, Candace M. Howard1, Corrado Minimo2, Cristiana Bellan3, Giovanna Carillo4, Luigi Califano5, Lorenzo Leoncini3, Antonio Giordano1,3 and Pier Paolo Claudio1

Authors' Affiliations: 1 Sbarro Institute for Cancer Research and Molecular Medicine, College of Science and Technology, Temple University; 2 Department of Pathology and Laboratory Medicine, College of Medicine, Drexel University, Philadelphia, Pennsylvania; 3 Department of Human Pathology and Oncology, University of Siena, Nuovo Policlinico "Le Scotte", Siena; 4 Dipartimento di Medicina di Laboratorio ed Anatomia Patologica, A.O. "Antonio Cardarelli"; and 5 Dipartimento di Scienze Odontostomatologiche e Maxillo-Facciali, Universita' degli Studi di Napoli "Federico II", Naples, Italy

Requests for reprints: Pier Paolo Claudio, Sbarro Institute for Cancer Research and Molecular Medicine, College of Science and Technology, Temple University, Biology-Life Sciences Building, Suite 333, 1900 North 12th Street, Philadelphia, PA 19122-6099. Phone: 215-204-9523; Fax: 215-204-9522; E-mail: claudio{at}temple.edu.


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
The retinoblastoma family consists of the tumor suppressor nuclear phosphoprotein pRb/p105 and related proteins p107 and pRb2/p130. Recent immunohistochemical studies of the retinoblastoma family of proteins in lung and endometrial cancer and choroidal melanomas show a tight inverse correlation between the histologic grading in the most aggressive tumor types and pRb2/p130 expression. This led us to investigate the role of pRb2/p130 in salivary tumors. We studied the expression of pRb2/p130, p107, E2F4, p27, and PcNA by immunohistochemistry in a panel of 44 salivary gland tumors. We found a direct correlation between the cytoplasmic expression of pRb2/p130 and tumor grading and the presence of metastasis that was highly statistically significant (P < 0.001). Additionally, increased cytoplasmic pRb2/p130 expression was significantly correlated with a decreased probability of survival (P < 0.001). Interestingly, p107 nuclear expression showed a strong direct correlation when compared with the same variables. pRb2/p130 showed the highest percentage of undetectable nuclear levels in the specimens examined and the tightest inverse correlation (P < 0.0001) with both the histologic grading and pCNA expression in malignant salivary tumors. Additionally, E2F4 showed an identical localization pattern as to that of pRb2/p130. These data suggests an important role for pRb2/p130 in the pathogenesis and progression of certain salivary gland cancers.

Key Words: Salivary gland tumors • Retinoblastoma • Cell cycle • Prognostic factor

Salivary gland tumors are relatively uncommon, comprising no more than 1% of all tumors and 3% of all the head and neck neoplasias. The highest incidence occurs between 30 and 60 years of age (1). In general, the incidence shows little variation between Europe and U.S., where major studies have been undertaken. However, exceptions occur in some populations. Inuit are a particularly high-risk group. In the non-White populations of the U.S. and Africa, females are affected more commonly than males (2.5/100,000 against 1.6/100,000, respectively; ref. 2). The parotid glands are by far the most commonly involved, and tumors at this site are approximately 10 times more common than those in either the submandibular glands or the minor glands taken as a group (3). In the White population of both Europe and the U.S., there is no significant sex predilection for salivary gland tumors. Benign tumors are more common than malignant growths, constituting about 75% of parotid tumors but accounting for <50% of the tumors of the other salivary glands (4).

Despite recent progress in molecular medicine, there is still a paucity of data regarding the involvement of cell cycle–regulatory proteins in the pathogenesis of head and neck tumors and in particular of the salivary glands. Accumulation of mutant p53 protein has been detected in both benign and malignant salivary gland tumors (5). Activation of c-myc and ras p21 proto-oncogenes have been recently depicted as important for the malignant transformation of salivary gland pleomorphic adenomas (6). A relatively high rate of mutations (loss of heterozygosity and microsatellite alterations) in the p53 gene have been detected in carcinomas and pleomorphic adenomas (58%), and slightly lower mutation frequencies of the Retinoblastoma (RB) gene (33%) have been reported (7).

The retinoblastoma gene family is composed of three members, the retinoblastoma gene (RB), which is one of the most studied tumor suppressor genes, and two related genes, RB2/p130 and p107. The proteins encoded by these genes, pRb2/p130 and p107, are structurally and functionally similar to pRb and play a pivotal role as negative regulators of cell proliferation (8). However, the three proteins exhibit unique growth-suppressive properties in specific cell lines, suggesting that although the different members of the retinoblastoma family may complement each other, they are not fully functionally redundant (9, 10). RB2/p130 maps to human chromosome 16q.12.2, an area that is frequently altered in several human neoplasias including breast, ovarian, hepatic, prostatic, and endometrial carcinomas, which suggests a possible role in the pathogenesis of several human cancers (11). We and others have recently reported that RB2/p130 is involved in the pathogenesis and progression of oral carcinomas (12–15), lung cancer (16–19), nasopharyngeal carcinoma (9, 20), lymphomas (21, 22), and is a strong predictor of clinical outcome in endometrial carcinomas (23), choroidal melanomas (24), as well as hepatocarcinomas (25).

The retinoblastoma family associates with different members of the E2F family. This interaction seems to be temporally modulated and varies between family members. Several sources of data suggest that pRb and p107 associate with distinct E2F species. The in vivo results show that E2F1, E2F2, E2F3, and E2F4 all form complexes with pRb, but E2F1-3 do not interact with p107 (26). E2F4 and E2F5 were both cloned for their ability to interact with pRb2/p130. E2F4 undergoes complex formation in vivo with pRb2/p130 during G0 and G1, and then later associates with p107 and pRb in late G1 and S phases as the levels of these regulators increase (27–30). However, the temporal order of complex formation seems to vary. For example, the binding of p107 to E2F is first detected at the G1-S boundary and remains stable throughout S phase (31, 32). On the other hand, pRb-E2F complexes are found in the G1 phase and then dissociate in late G1 (33, 34). The main form of E2F detected in the G0-G1 phases in primary mouse fibroblasts is E2F bound to pRb2/p130, which is then replaced by p107-E2F complexes in late G1 (35). Therefore, pRb2/p130 is the major negative modulator of E2F activity in quiescent cells. Similar to the case for pRb and E2F1, p107 and pRb2/p130 through complex formation with E2F4 stabilize the protein and protect E2F4 from targeted proteolysis by the ubiquitin-proteasome pathway. Stabilization of the p107- and pRb2/p130-E2F4 complexes is thought to contribute to maintaining a state of active transcriptional repression in quiescent cells (36). Unlike E2F1, -2, or -3, which are constitutively nuclear, E2F4 and E2F5 lack a nuclear localization signal. The subcellular localization of E2F4 is regulated in a cell cycle–dependent manner with high nuclear/cytoplasmic ratios in G0 and early G1 with a progressive increase in cytoplasmic E2F4 as the cells proceed toward S phase (37). Cotransfection studies indicate that cellular compartmentalization of E2F4 and E2F5 becomes nuclear by complex formation with p107, pRb2/p130, DP-2, and/or DP-3{delta} (37–39). E2F4 nuclear accumulation induced by pRb2/p130 and p107 correlates with cell growth arrest, whereas overexpression of E2F4 with DP-3{delta} stimulates re-entry into the cell cycle in previously quiescent cells. This E2F4/DP-3{delta} reactivation of the cell cycle, however, is efficiently counteracted by overexpression of either p107 or pRb2/p130 (38). Because of the paucity of molecular data on salivary gland tumors and all head and neck tumors in general, we decided to screen a panel of 44 tumors of the salivary glands by immunohistochemistry for the expression pattern of pRB2/p130, p107, E2F4, p27, and pCNA. The current study was undertaken to investigate whether the expression of the retinoblastoma-related proteins p107 and pRb2/p130 are predictors of clinical behavior in salivary gland tumors. We studied a panel of patients with different tumoral disease of the parotid gland that did not receive radiation or chemotherapy prior to surgery.

We have evaluated the relationship between the proteins' expression and clinicopathologic variables such as age, gender, smoking, and alcohol consumption, grading, histotype, and lymph node metastasis. No statistically significant correlations were found between age, sex, smoking, alcohol consumption, and the proteins' staining. Lack of nuclear expression and conversely, high cytoplasmic expression of pRb2/p130 indicated an increased likelihood of metastasis and a decreased chance of survival. Therefore, the evaluation of the expression pattern of the tumor suppressor gene RB2/p130 may serve as a new prognostic factor in salivary glands tumors.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Patient populations and clinical data. Paraffin-embedded sections of salivary gland tumors were obtained from patients who underwent surgical resection as first-line of treatment at the Department of Maxillo-Facial Surgery, University of Naples "Federico II", Naples, Italy. Patients signed an informed consent for the study which was reviewed by the Institutional Review Board. Staging was carried out according to the tumor-node-metastasis classification, and the tumors were graded as well (G1), intermediate (G2), and poorly (G3) differentiated. Three experienced pathologists (C. Minimo, L. Leoncini, and C. Bellan) confirmed blindly and independently the histologic diagnosis of each salivary lesion. Adjacent uninvolved salivary gland tissues were also present for evaluation in the examined sections. We examined a total of 13 squamocellular carcinomas, 4 adenocarcinomas, 2 carcinomas in pleomorphic adenoma, 3 adenoid-cystic carcinomas, 10 Warthin's tumors, and 12 pleomorphic adenomas. Ten normal salivary tissues were used as control in addition to the normal tissue often found in the studied samples. Only patients with primary salivary gland tumors who had not undergone any previous irradiation or chemotherapeutic treatment were included in the study.

Immunohistochemistry. A total of 44 formalin-fixed and paraffin-embedded tumor tissue samples were processed (Table 1). Sections of each specimen were cut at 3 µm, mounted on glass and dried overnight at 37°C. All sections were dewaxed, rehydrated, and quenched in 0.5% hydrogen peroxide and microwave pretreated in 10 mmol/L citrate buffer (pH 6.0; 15 minutes at 650 W for p107, 18 minutes at 650 W for pRB2/p130). After blocking with normal serum for 1 hour at room temperature, our rabbit polyclonal antibody against p107 was incubated with tissue sections at a 1:1,000 dilution at room temperature for 4 hours. Our affinity-purified rabbit polyclonal immune serum against pRB2/p130 was used instead at 4°C overnight at a 1:50 dilution. The mouse monoclonal antibody against pCNA (clone PC10, DAKO, Carpinteria, CA) was incubated with tissue sections at a 1:200 dilution at room temperature for 2 hours. The polyclonal antibodies against p27 and E2F4 (C-20; Santa Cruz, Santa Cruz, CA) were incubated with tissue sections at 1:100 dilution at 4°C overnight.


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  		Table 1. Tumor tissue samples of patient cohort

 
Negative controls were produced by substituting the primary antibodies with preimmune serum. The positive immunostaining of infiltrating lymphocytes represented an internal positive control for preservation of antigenicity in the sections examined. All slides were processed by the avidin-biotin complex method (Vector Laboratories, Burlingame, CA). Diaminobenzidine was used as the final chromogen, and Gill's hematoxylin was used for counterstaining.

p107, pRB2/p130, E2F4, p27, and PcNA scoring. Three pathologists (C. Minimo, L. Leoncini, and C. Bellan) blindly and independently evaluated each specimen and scored the immunostaining for the percentage of positive nuclei. The level of concordance, expressed as the percentage of agreement between the observers, was 93.2% (41 of 44 specimens). In the remaining three samples, the score was obtained from the re-evaluation of the slides by the three observers. At least 20 high-power fields were chosen randomly and 2,000 cells were counted. Tumor sections were considered negative (score 0) if staining was absent or present in <25% of tumor cells. A score of 1 was given when 25% to 50% of the cells were positive to the reaction. A score of 2 was given when 50% to 75% or >75% of the cells were positive to the reaction, respectively.

Follow-up and evaluation of results. After completing the treatment, patients were seen 1 month after surgery and then at 3 and 6 months and a year from the intervention, for the first year. Then, patients were seen every 6 months every year during the second, third, fourth, and fifth year. Recurrence was considered as any documented relapse of the tumor either locally or dissemination to lymph nodes. Disease-free interval was calculated from the date of the operation. Patients who recurred within 3 months from the date of the operation were not considered free of disease and were therefore excluded from the disease-free analysis, but not from the survival calculation. Eight patients died from causes other than salivary gland cancer (18.1%) and were considered as lost to follow-up and therefore their survival times were censored at the date of death. Follow-up data were available for all the patients, with a median of 48 months (range 36-60 months). Disease-free interval and time to salivary gland tumor–related death were the endpoints of the study.

Statistical analysis. Fisher's exact test was used to evaluate the association between pRb2/p130, p107, p27, and E2F4 expression and the other prognostic variables (40, 41). Correlation between variables was determined using the Spearman correlation rank test and the Wilcoxon signed rank test. Disease-free interval and disease-specific survival were calculated according to the Kaplan-Meier method (42) and evaluated by the log-rank test (43). Univariate Cox analysis was used to assess the effect of each prognostic variable on disease-free interval and survival. A multivariate analysis (Cox proportional-hazards regression, with forward selection of variables; ref. 44) was done to estimate which of the possible risk factors yielded independent prognostic information. Data analysis was done with the SPSS statistical package, release 5.0.1 (SPSS, Inc., Chicago, IL). Results were considered statistically significant when the P value was <0.05.


   Results
Top
 Abstract
 Materials and Methods
 Results
Discussion
 References
 
pRB2/p130, p107, E2F4, p27, and PcNA protein expression in salivary gland tumors. The expression of pRB2/p130, p107, E2F4, p27, and pCNA in salivary tissues was determined by immunohistochemistry. pRb2/p130 showed a clear nuclear immunoreactivity in normal salivary glands. A variable level of pRb2/p130 nuclear expression between 0 and 2 was found among all the samples examined. Considering the whole cohort of the cases studied, loss of nuclear expression of pRB2/p130 (score 0), was found in 26 cases (59.1%), 15 cases (34.1%) showed a nuclear score of 1 (25-50% positivity), and three cases (6.8%) had a score of 2 (50-75% positivity). Only three cases (6.8%) showed <50% of the cells positive for pRb2/p130 cytoplasmic staining (score 0 or 1), 14 cases (34%) had a score of 2 (50-75% positivity), and 27 other cases (61.4%) or had a score of 3 (>75% positivity).

Interestingly, 100% of the 22 malignant specimens revealed a cytoplasmic expression pattern of pRb2/p130, and in 19 cases (86.4%), the nuclear score was zero (<25% positivity), whereas in 20 of those cases (90.1%), the cytoplasmic score was between 2 and 3 (50-75% and >75%, respectively; Fig. 1A-D). p107 showed a prevalent nuclear staining in actively proliferating tumors (Fig. 1E-H).



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  		Fig. 1. Immunohistochemical analysis of pRb2/p130 (A-D) and p107 (E-H) in salivary gland tumors. A, pRb2/p130 expression in normal salivary gland. B, pRb2/p130 expression in Warthin's tumor. C, pRb2/p130 expression in pleomorphic adenoma. D, cytoplasmic pRb2/p130 expression in high-grade salivary gland carcinoma. E, p107 expression in normal salivary gland. F, p107 expression in Warthin's tumor. G, p107 expression in pleomorphic adenoma H, nuclear p107 expression in high-grade salivary gland carcinoma.

 
E2F4 showed an identical localization pattern as pRb2/p130. In particular, squamous cell carcinomas showed a high E2F4 cytoplasmic score (>75% cellular positivity). Pleomorphic adenomas showed a nuclear E2F4 staining >50% cellular positivity in 60% of epithelial cells and in 50% of the stromal cellular component, respectively. Whartin's tumors, conversely, exhibited a cytoplasmic E2F4 staining in 87% of the cases with a cellular positivity higher than 75% and an absent or a low nuclear staining (score 0-1). p27 showed intense nuclear staining in normal salivary glands and in well-differentiated carcinomas, whereas it showed absent immunoreactivity in poorly differentiated carcinomas (Fig. 2A-D). pCNA expression was strongly positive in less differentiated tumors (Fig. 2E-H).



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  		Fig. 2. Immunohistochemical analysis of p27 (A-D) and pCNA (E-H) in salivary gland tumors. A, p27 expression in normal salivary gland. B, p27 expression in Warthin's tumor. C, p27 expression in pleomorphic adenoma. D, p27 expression in high-grade salivary gland carcinoma. E, pCNA expression in normal salivary gland. F, pCNA expression in Warthin's tumor. G, pCNA expression in pleomorphic adenoma. H, pCNA expression in high-grade salivary gland carcinoma.

 
Correlation of pRB2/p130, p107, E2F4, p27, and PcNA expression with clinicopathologic variables. The relationship between the expression of pRB2/p130, p107, E2F4, p27, and pCNA with a series of clinicopathologic variables (age, grading, histotype, smoke, and alcohol consumption, lymph nodal metastasis, and outcome) was investigated in 44 salivary gland tumors (for patients' cohort see Table 1).

The median age of the patients' was 49 years (range 26-66), the median age of the patients affected by malignant tumors was 52 years (range 42-66), and the median age of the patients affected by benign tumors was 45 years (range 26-62).

Comparing the expression pattern of pRb2/p130 with tumor grade, we found a direct correlation between cytoplasmic expression and tumor malignancy (Fig. 3). In fact, in all the malignant cases classified as well-differentiated (G1), the cytoplasmic score was always ≤2, whereas the poorly differentiated carcinomas (G3) showed a score of 3 (>75% positivity). Additionally, among the cases with a pRb2/p130 cytoplasmic score of 3 (>75% positivity; 7 cases out of 10), 70% were diagnosed as poorly differentiated (G3). The direct correlation between pRb2/p130 cytoplasmic expression, and neoplastic grading was highly statistically significant (P < 0.0001). Other relevant correlations are those between pRb2/p130 cytoplasmic expression pattern and lymph nodal metastasis and survival (P < 0.001; Figs. 4 and 5). In fact, among the cases with a pRb2/p130 cytoplasmic score of 2 (50-75% positivity; nine cases), three of the cases (33%) had lymph nodal positivity, and among those with a cytoplasmic score >2 (>75% positivity; 10 cases), seven of the cases (70%) had lymph nodal positivity. pRb2/p130 (P < 0.0001) also showed the highest percentage of undetectable nuclear levels in the specimens examined and the tightest inverse correlation (P < 0.0001), with both the histologic grading and pCNA expression in malignant salivary tumors. This, in addition to the reduced survival of patients with pRb2/p130 cytoplasmic staining >75% (score 3; Fig. 5), reinforces the prognostic value of the pRb2/p130 cytoplasmic localization which has become an index not only of malignancy but also of aggressivity.



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  		Fig. 3. Graphic representation of cytoplasmic pRb2/p130 staining and tumor grade. High cytoplasmic immunoreactivity of pRb2/p130 directly correlates with the tumor grade. Malignant cases classified as well-differentiated (G1), showed a cytoplasmic score equal or lower than two, whereas the poorly differentiated carcinomas (G3) showed a score of 3 (>75% positivity).

 


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  		Fig. 4. Graphic representation of cytoplasmic pRb2/p130 staining and nodal metastasis in malignant salivary gland tumors. High cytoplasmic immunoreactivity of pRb2/p130 directly correlates with the presence of lymphonodal metastasis. Cases with cytoplasmic scoring of 2 or higher showed lymphonodal positivity.

 


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  		Fig. 5. Graphic representation of cytoplasmic pRb2/p130 staining and patient survival in malignant salivary gland tumors. High cytoplasmic immunoreactivity correlated with an increased incidence of death. Cases with cytoplasmic scoring of 3 were those with a higher number of salivary gland tumors related to deaths.

 
p107 instead, showed a positive correlation between nuclear expression pattern and grading. In fact, in the malignant neoplasias we examined, p107 was expressed mostly in the nucleus in 20 cases (90%), and in a relatively high number of specimens (50%) with a high score (2-3). Another statistically significant data is that 100% of the pleomorphic adenomas showed exclusive p107 nuclear expression.

The data that p107 is expressed in the nucleus more than in the cytoplasm in more actively proliferating cells and that this inversely correlates, in the same cases, with a cytoplasmic expression of pRb2/p130 is in accordance with previous reports (21, 22, 45).

Among the benign neoplasias of the salivary glands we examined, the comparison of the histologic data with the expression pattern of pRb2/p130 showed significance. In fact, Warthin's tumors, in general, showed lower nuclear expression of pRb2/p130 when compared with that of pleomorphic adenomas.

Interestingly, the expression pattern of p107 showed a peculiar distribution linked to the histotypes. In Warthin's tumors, p107 was mostly expressed in the cytoplasm, whereas in pleomorphic adenomas, its expression levels were shifted to the nuclear compartment. We do not currently have an explanation for this peculiar expression pattern of p107, and we are presently investigating the differential role of p107 and pRb2/p130 in salivary benign tumors.

E2F4 showed an identical localization pattern as pRb2/p130. In particular, squamous cell carcinomas showed a high E2F4 cytoplasmic score (>75% of cellular positivity). Pleomorphic adenomas showed a nuclear E2F4-positive staining >50% in 60% of epithelial cells and in 50% of the stromal cellular components, respectively. Whartin's tumors, conversely, exhibited a cytoplasmic E2F4 staining in 87% of the cases (score 3) and an absent or a low nuclear staining (score 0-1). Most importantly, the E2F4 expression was mostly limited to the cytoplasmic fraction in those tumors that showed a cytoplasmic staining for pRb2/p130. Pleomorphic adenomas that, as aforementioned, have almost an exclusive pRb2/p130 nuclear localization showed nuclear E2F4 staining in 90% of the cases. Conversely, Warthin's tumors that showed a high cytoplasmic expression of pRb2/p130, exhibited a high E2F4 cytoplasmic immunoreactivity (score 3) in 87% of the cases. Finally, a positive correlation was found between p27 nuclear positivity of expression and tumor differentiation.


   Discussion
Top
 Abstract
 Materials and Methods
 Results
 Discussion
References
 
Recently, Etges et al. (46) studied the immunohistochemical behavior of proteins involved in the retinoblastoma (pRb/p105) pathway in a panel of salivary gland tumors and matched normal tissues. The authors found that benign and malignant tumors expressed retinoblastoma pathway proteins such as cyclin D1, cyclin-dependent kinase (CDK)-4, the retinoblastoma protein (pRb/p105), CDK inhibitor p16 and the transcription factor E2F1 differently from normal salivary glands. The authors' findings suggest that the pRb/p105 pathway deregulation in salivary gland neoplasms is unrelated to their biological behavior (46). Because of the paucity of molecular data on the expression of the retinoblastoma family members (pRb/p105, p107, and pRb2/p130) in salivary gland tumors and head and neck tumors in general, we decided to investigate whether the expression of the other two retinoblastoma family proteins, p107 and pRb2/p130, are predictors of clinical behavior in salivary gland tumors. Therefore, we screened a panel of 44 tumors of the salivary glands by immunohistochemistry for the expression pattern of pRB2/p130, p107, E2F4, p27, and pCNA. The patients studied were affected by a tumoral disease of the parotid gland that did not receive radiation or chemotherapy prior to surgery.

pCNA, an auxiliary protein of the DNA polymerase {delta}, is a proliferation-associated marker. Its maximal expression peaks in late G1 and S phase of the cell cycle (47). pCNA has been used as a proliferation marker in different neoplasms in relation to clinical behavior (48). Our results confirm previous studies in salivary gland tumors in which high levels of pCNA expression have been associated with biologically aggressive tumors (49–51).

The retinoblastoma gene family is composed of three members, the retinoblastoma gene (RB), which is one of the most studied tumor suppressor genes, and two related genes, RB2/p130 and p107. The proteins encoded by these genes, pRb2/p130 and p107 are structurally and functionally similar to pRb and play a pivotal role as negative regulators of cell proliferation (8).

We, and others have recently reported that RB2/p130 is involved in the pathogenesis and progression of lung cancer (16–19), nasopharyngeal carcinoma (9, 20), and lymphomas (21, 22), and is a strong predictor of clinical outcome in endometrial carcinomas (23), choroidal melanomas (24) as well as hepatocarcinomas (25). Additionally, recent studies described the expression of pRb2/p130 related to the degree of differentiation of oral squamous cell carcinoma as that of Rb/p105 (12, 13, 15). According to Tanaka et al. 12, pRb2/p130 expression was shown to be a better marker of tumor differentiation with respect to Rb/p105 in oral squamous cell carcinoma.

Functional studies of p107 and pRb2/p130 indicated that although the RB family members may be able to complement each other, these proteins are not fully functionally redundant (9, 10). In fact, p107 interacts with E2F4 and E2F5, as does pRb2/p130; however, the constant presence of the E2F/p107/cyclin A/CDK2 complex in proliferating cells (52) suggests that p107, rather than acting exclusively as a tumor suppressor protein (10), may also play a positive role in cellular proliferation (53). The differential expression of p107 and pRb2/p130 in malignant salivary glands presented here are in accordance with these experimental data and support the concept of their different function in cellular growth control.

A model for pRb2/p130 and p107 control during cellular proliferation has been proposed (53) in which the E2F-pRb2/p130 complex in quiescent cells, is responsible for the active repression of a number of cellular promoters, including those of E2F1, E2F2, and p107 (8, 53, 54). In order to progress in the cell cycle, pRb2/p130 is phosphorylated by G1 CDKs and then degraded through a proteasome-dependent mechanism resulting in the de-repression of a variety of genes including p107 (36, 54). p107 accumulates and interacts with free E2F4 and E2F5 released from the pRb2/p130 complex and also associates with cyclin A/CDK2 (31). Therefore, according to these data, the amount of cells expressing p107 and pRb2/p130 should be inversely related.

Alterations in the p107 gene have also been identified in a limited number of human hematologic malignancies (55). Altered pRB2/p130 expression has been reported in several tumors, including oral carcinomas, primary lung cancer, endometrial carcinomas, prostate carcinomas, lymphomas, choroidal melanomas, and hepatocarcinomas, and has been correlated with a poor clinical outcome (12, 13, 15, 18, 19, 23–25, 45, 56).

Additionally, pRb2/p130 has been shown to elicit its tumor-suppressive capacity in vitro and in vivo using a tetracycline-dependent expression system as well as retroviral-mediated delivery (17, 57). These results and the idea that Rb2/p130 could be used in the future as gene therapeutic tool, led us to investigate the role of pRb2/p130 in salivary tumors.

We found a direct correlation between the cytoplasmic expression of pRb2/p130 and tumor grading (degree of malignant potential; P < 0.0001) and the presence of metastasis (P < 0.001) which was highly statistically significant. A direct correlation was also found comparing the pRb2/p130 cytoplasmic expression and the patients' poor survival that was statistically significant (P < 0.001). Conversely, p107 showed a strong direct correlation when compared with the same variables. This phenomenon was particularly evident for pRb2/p130 nuclear expression that had the highest percentage of undetectable levels in the specimens examined and the tightest inverse correlation with both the histologic grading and pCNA expression in the most aggressive tumor types (G2-G3). These data suggest an important role for pRb2/p130 in the pathogenesis and progression of certain salivary gland cancers and indicate that the tumor suppressor gene RB2/p130 may be considered a new prognostic marker for salivary gland neoplasms. However, additional studies of the pRb2/p130 nuclear-cytoplasmic expression pattern on a greater number and more homogeneous number of salivary gland tumors are needed to confirm the clinical significance the correlation between histologic grading, pCNA and nuclear-cytoplasmic pRb2/p130 expressions that together could be new biomarkers in salivary gland tumors.


   Acknowledgments
 
We thank Dr. Antonio Farina for his precious help in the statistical analysis of the results. G. Russo acknowledges the Ph.D. program "Diagnostic, quantitative, and molecular pathology" of the University of Siena, Italy.


   Footnotes
 
Grant support: W.W. Smith Charitable Trust grant to P.P. Claudio and by NIH grant to A. Giordano.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 12/ 6/04; revised 1/17/05; accepted 2/ 9/05.


   References
Top
 Abstract
 Materials and Methods
 Results
 Discussion
 References
 

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