Role of Retinoblastoma Protein Family (Rb/P105 And Rb2/P130) Expression in the Hystopathological Classification of Borderline Ovarian Tumors

Role of Retinoblastoma Protein Family (Rb/P105 And Rb2/P130) Expression in the Hystopathological Classification of Borderline Ovarian Tumors | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Role of Retinoblastoma Protein Family (Rb/P105 And Rb2/P130) Expression in the Hystopathological Classification of Borderline Ovarian Tumors Valeria Masciullo, Paola Valdivieso, Giulia Amadio, Angela Santoro, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-48452/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Borderline ovarian tumors (BOT) are uncommon but not rare epithelial ovarian neoplasms, intermediate between benign and malignant categories. Emerging knowledge supports the notion that subtypes of borderline ovarian tumors comprise distinct biologic, pathogenetic, and molecular entities, precluding a single unifying concept for BOT. Therefore, the identification of valuable markers for the diagnosis and classification of these tumors is in need. Among the molecular candidates, the Retinoblastoma (Rb) family members Rb/p105 and Rb2/p130 seem to play a pivotal role in ovarian cancer. In particular, Rb/p105, when in the unphosphorylated form, acts as a growth suppressor and plays a pivotal role in the negative control of the cell cycle and in tumor progression; whereas, the phosphorylated form (p-pRB) activates genic transcription and cellular proliferation. While Rb/p105 is ubiquitously confined to the nuclei of cycling and quiescent cells, Rb2/p130 activity is also regulated by intracellular localization. According to this premise, Rb family members could represent a novel marker in diagnosis and classification risk for patients with borderline ovarian tumors (BOT). Aims In this study, we evaluated the immunohistochemical expression and subcellular localization of proteins of the retinoblastoma (Rb) gene family: Rb/p105 and Rb2/p130 in 65 ovarian borderline tumors (26 serous, 19 sero-mucinous and 20 mucinous subtypes). Results Statistically significant differences were found in nuclear and cytoplasmic expression of Rb/p105 and Rb2/p130 according to different examined histotypes. In detail, the nuclear expression of Rb/p105 and Rb2/p130 was more frequently detected in serous (84.6%) than sero-mucinous (42.1%) and mucinous (50%) types. Conversely, the cytoplasmic expression of Rb2/p130 was not detected in serous tumors and frequently observed in mucinous subtypes (80%). Conclusions Our findings suggest that Rb proteins do not play a key role in the tumor progression of serous borderline tumors since they are always located in the nucleus and no cases showed a cytoplasmic localization. By contrast, the observed higher cytoplasmic expression of Rb2/p130 in mucinous BOTs (intestinal) types, is suggestive of Rb proteins involvement in the cancerogenesis pathway of mucinous ovarian tumors. Our results also suggest that mucinous BOTs of intestinal type, exhibiting low nuclear and high cytoplasmic levels of Rb2/p130 might potentially be considered a high risk category of malignant evolution. Further studies on larger series are needed in order to clarify how BOTs could be stratified in different prognostic groups according to their Rb proteins immunohistochemical profile. Animal Science Retinoblastoma family proteins borderline ovarian tumors pRb/p105 pRb2/p130 diagnosis Figures Figure 1 Figure 2 Figure 3 Figure 4 Background Borderline ovarian tumors (BOTs) represent one of the controversial topics in gynecologic pathology [ 1 ]. They are a heterogeneous group of tumors that account for 10–20% of all ovarian epithelial neoplasms. The most common BOT hystotypes are serous (50%) and mucinous (45%) with less common subtypes (5%) including sero-mucinous, endometrioid, clear cell, and borderline Brenner tumors [ 2 ]. The 97% of all stages of BOT have a good prognosis with a mean 10-years survival [ 4 ], although recurrences and malignant transformation can occur in a very small proportion of cases [ 5 ]. In contrast to serous BOTs, that are rarely characterized by evolution in a low grade serous carcinoma whereas are often associated to peritoneal implants and relapses [ 5 ], mucinous carcinoma frequently develops from benign and borderline mucinous tumors [ 6 , 7 ]. Similarly sero-mucinous BOTs are often the land of endometrioid or clear cell carcinoma and usually represent a morphologic continuum in the middle of benign and malignant counterparts. BOTs often occur in young women, however the absence of stromal invasion warrant a better prognosis compared to ovarian carcinoma [ 8 , 9 ]. Neverthless, since the standard treatment of BOTs is usually surgery, the fertility of these women may be affected [ 10 ]. Identifying the genetic background for diagnosis and prognosis should avoid a radical resection and help in developing new targeted therapies, especially in younger woman with desire of childbearing. Thus, a better understanding of the clinical phenotype and pathogenesis of BOTs would contribute to their earlier detection and is essential for development of more effective treatments. Previous studies support the idea that the serous and mucinous BOT have distinct carcinogenic pathways. For example, the expression of p21 and MDM2 differs between mucinous and serous forms [ 11 ]. In other studies, a higher rate of p53 mutation was observed in mucinous compared to serous BOTs and p21 and bcl-2 overexpression appeared specific to serous forms and different between serous benign, serous BOTs and serous carcinoma [ 12 – 14 ]. The retinoblastoma gene family consists of three members and their products are: Rb/p105, Rb2/p130 and RbL1/p107, together known as “pocket proteins” family [ 15 , 16 ]. Their most important target is the E2F-family of transcription factors which control the expression of genes that mediate G1-S transition [ 15 , 16 ]. The localization of these proteins into the nucleus or around the nuclear membrane has been shown at the molecular level during the different phases of the cell cycle [ 17 , 18 ]. In detail, during the cell progression through the S into the G2/M phases of division cycle, pRB undergoes phosphorylation, while in the late M phase, pRB is rapidly dephosphorylated. When pRB is in the unphosphorylated form, it acts as a growth suppressor by repressing transcription of E2F. By contrast, the phosphorylated pRB status (p-pRB) leads to activation of E2F-responsive genes and entry into the S phase. While Rb/p105 is ubiquitously confined to the nuclei of cycling and quiescent cells, Rb2/p130 activity is also regulated by intracellular localization. The phosphorylation status of Rb2/p130 itself, therefore, is important in the regulation of cell cycle [ 19 ]. The hyperphosphorylated form of pRb2/p130 is cytoplasmic and typical of cells progressing into G1 phase [ 20 ]. Alteration of Rb family members is frequently involved in gynaecological cancers [ 21 – 23 ]. We previously showed that the loss of Rb2/p130 or its cytoplasmic expression occurs in 40% of ovarian tumors and is inversely correlated with tumor grade [ 24 ]. However, little is known about Rb proteins expression in borderline ovarian tumors. In this study, we utilized immunohistochemistry to evaluate the expression pR/p105 and pRb2/p130 family members in a large, single-institution, series of mucinous, sero-mucinous and serous BOTs. Materials And Methods Data collection Sixty-five ovarian BOTs were retrospectively collected from patients who underwent salpingo-oophorectomy for ovarian cancer in the Division of Gynaecologic Oncology of the Fondazione Policlinico Universitario A.Gemelli IRCSS, Rome, Italy between 2010 and 2016; all selected patients did not receive chemotherapy or radiotherapy before surgical enucleation. All the subjects gave a written informed consent before enrollment. Twenty specimens were defined as mucinous BOTs, 19 were classified as sero-mucinous, whereas 26 cases were classified as serous BOTs. Histological classification of tumors was carried out according to the WHO system, and disease staging was established according to the International Federation of Gynaecologist and Obstetricians (FIGO) criteria. Immunohistochemistry After surgical resection, tissues were immediately fixed in 10% formalin and then paraffin-embedded for immunohistochemical analysis. The immunostaining was performed using a streptavidin-biotin complex immuno peroxidase method (DakoCytomation). Detection for the retinoblastoma gene family members was performed using purified mouse anti-human retinoblastoma protein (Rb) monoclonal antibody (BD Pharmigen) diluted 1:50 and mouse monoclonal antibody Rb2 p130 (clone 130-P215; Novus Biologicals, Inc.) diluted 1:25. Paraffin blocks of each specimen were sectioned at 3 µm, mounted on slide and dried overnight at 37 °C. All sections were dewaxed in xylene and dehydrated in descending graded alcohols to Phosphate-Buffered Saline (PBS; pH 7.4). Antigen retrieval was performed by microwaves in 10 mM Citrate buffer (pH 6), at 750 W for 10 min (two cycles of 5 min each), followed by cooling at room temperature for at least 20 min prior to incubation with the antibodies. Sections were treated with 0.4% H 2 O 2 methanol solution (15 min at room temperature to inhibit endogenous peroxidase activity), quickly rinsed in water and then in PBS. Sections were then placed in a humidified chamber and incubated with primary antibody at room temperature for 40 min. The sections were then washed in PBS (two times for 5 min each). Antigen detection was carried out by exposure to a biotinylated universal secondary antibody for 10 min followed by a streptavidin-peroxidase complex working solution for 10 min. After another PBS wash, the antigen antibody complex was visualized by staining with chromogen 3,3’-diaminobenzidine/ tetrachloride solution (DAB, Vector) for 5 min. The sections were rinsed in deionized water; cell nuclei were counterstained with hematoxylin and dehydrated in graded alcohols followed by xylene. Specimens of human colon cancer, follicolar cyst and falloppian tube served as positive controls for pRb and pRb2, respectively. For negative control, slides were simultaneously incubated with PBS in the absence of the primary antibody. The results were independently reviewed by three experienced pathologists (GFZ, GA, AS), who were blinded to clinical outcome at the time of evaluation. Discrepancies in the evaluation (< 10% of cases) were resolved by re-observation of the cases using a multi-headed microscope. Quantitative scoring of protein expression was based on the staining intensity and the percentage of positive cells as follows: negative (0%); lower positive (1–30%) or upper positive (> 30%) cells. A cut-off of 30% was considered statistically significant and, therefore, functionally operative. Statistical Analysis The associations between Rb proteins staining and other clinic-pathological parameters were analysed using contingency table methods and tested for significance using the Fisher’s exact χ2 test. All calculation were performed using the Statistical Package for Social Science (SPSS 17.0 software, Chicago, IL) and the result was considered statistically significant when the P value was less or equal to 0.05. Results Clinico-pathological features The series included 20 mucinous, 19 sero-mucinous and 26 serous BOTs. The mean and median age of the patients were 44.7 and 44 years (range 20–72), respectively. All mucinous and sero-mucinous selected tumors (39) were Stage 1, without evidence of implants or recurrences, whereas serous tumors included stage 1 (n = 20), stage 2 (n = 1) and stage 3 (n = 5) cases and follow-up data were available for all 65 patients. Five serous BOTs were classified as BOTs with microinvasive foci. One mucinous BOT showed foci of intraepithelial carcinoma and an area of malignant invasive mucinous carcinoma with expansive pattern of growth. Fifty-nine cases were limited to the ovary, without peritoneal implants and 6 cases (all serous BOTs) were associated to peritoneal implants (only 1 of invasive type). Sixty-one cases did not relapse; the remaining 4 developed peritoneal recurrences. By the time this study was undertaken, no patients had died of the disease. Clinico-pathologic characteristics of patients are summarized in Table 1 . The expression levels of Rb/p105 and Rb2/p130 were determined by immunohistochemistry. Table 1 Clinical data of the patients with BOTs HISTOLOGIC TYPES Serous subtype* Sero-Mucinous subtype Mucinous subtype** N° (% of Cases) 26 (40.0) 19 (29.2) 20 (30.8) STAGE 1 2 3 N° (% of Cases) 59 (90.8) 1 (1.5) 5 (7.7) IMPLANTS Not Implants Implants without invasion Implants with invasion N° (% of Cases) 59 (90.8) 5 (7.7) 1 (1.5) RECURRENCES No Yes N° (% of Cases) 61 (93.8) 4 (6.2) CLINICAL OUTCOME Dead Alive N° (% of Cases) 0 65 (100) * 5 serous BOTs showed micro-invasive foci ** 1 mucinous BOT showed foci of intraepithelial carcinoma and an area of invasive mucinous carcinoma with expansive pattern of growth Correlation of Rb/p105 with clinic-pathological parameters in BOTs. The expression of Rb/p105 according to clinico-pathological parameters is shown in Table 2 . Notably, the immunoreactivity for Rb/p105 was only nuclear in our series. Moreover, the nuclear Rb staining in intestinal type mucinous BOTs tended to concentrate at the bases of the papillary projections (Fig. 1 ). Table 2 Nuclear Distribution of Rb/p105-negative and Rb/p105-positive cases according to tumoral characteristics Rb/p105 Nuclear Total N° Rb/p105 negative (0%) N° (%) Rb/p105 positive (1–30%) N° (%) Rb/p105 positive (> 30%) N° (%) P HISTOLOGIC TYPES 65 25 (38.4) 33 (50.8) 7 (10.8) Serous Sero-Mucinous subtype Mucinous subtype 26 19 20 4 11 (57.9) 10 (50.0) 22 (84.6) 6 (31.6) 5 (25.0) 0 (15.4.) 2 (10.5) 5 (25.0)) < 0.0001 STAGE 1 2 + 3 59 6 25 (42.3) 0 27 (45.8) 6 (100) 7 (11.9) 0 0.04 IMPLANTS Absent Present 59 6 24 (41.4) 1 (14.3) 27 (46.5) 6 (85.7) 7 (12.1) (0) NS RECURRENCES Absent Present 61 4 24 (39.4) 1 (25.0) 31 (50.8) 2 (50.0) 6 (9.8) 1 (25.0) NS Numbers in parentheses represent the percentage of specimens achieving that particular score. NS, not significant. The nuclear expression of Rb/p105 was observed in 40 (61.6%) out of the 65 patients, whereas it was not detectable in the remaining 25 cases (38.4%). The expression of nuclear Rb/p105 was more frequently detected in serous (22 cases; 84.6%) than in serous mucinous (8/19, 42.1%) and mucinous (10/20, 50%) types, and this difference was statistically significant (P < 0.0001). Positive staining was observed in 34 (57.6%) out of 59 stage 1 and in all 6 (100%) stage 2/3 cases and this difference was slightly significant (p = 0.04). No statistically significant correlation was observed between the nuclear expression of Rb/p105, implants and recurrences (Table 2 ). Correlation of Rb2/p130 expression with clinico-pathological parameters in BOTs Nuclear Rb2/p130 expression was detected in 33 (50.8%) of the 65 cases whereas it was absent in the remaining 32 cases (40.2%) (Fig. 2 ). The expression of nuclear Rb2/p130 was more frequent in serous (21/26, 80.8%) than in sero-mucinous (10/19, 52.6%) and intestinal (2/20, 1.0%) types, and these differences were statistically significant (P < 0.0001). Positive staining was observed in 27 (45.7%) out of the 59 stage 1 and in all 6 (100%) stage 2/3 cases and this difference was significant (p = 0.03). No statistically significant correlation was observed between the nuclear expression of Rb2/p30, implants and recurrences ( Table 3A ) Table 3 (A) Nuclear d istribution of Rb2/p130-negative and Rb2/p130-positive cases according to tumoral characteristics Rb2/p130 Nuclear Total N° Rb2/p130 negative (0%) N° (%) Rb2/p130 positive (1-30%) N° (%) Rb2/p130 positive(>30%) N° (%) P HISTOLOGIC TYPES Serous Sero-Mucinous subtype Mucinous subtype 26 19 20 5 (19.2) 9 (47.4) 18 (90.0) 7 (26.9) 5 (26.3) 2(10.0) 14 (53.9) 5 (26.3) 0 <0.0001 STAGE 1 2+3 59 6 32 (54.3) 0 12 (20.3) 2 (33.3) 15 (25.4) 4 (66.7) 0.03 IMPLANTS Absent Present 59 6 31 (53.4) 1 (14.3) 12 (20.7) 2 (28.6) 15 (25.9) 4 (57.1) NS RECURRENCES Absent Present 61 4 30 (49.2) 2 (50.0) 12 (19.7) 2 (50.0) 19 (31.1) 0 NS Numbers in parentheses represent the percentage of specimens achieving that particular score. NS, not significant Table 3 (B) Cytoplasmic distribution of Rb2/p130-negative and Rb2/p130-positive cases according to histologic types Rb2/p130 Cytoplasmatic Total N° Rb2/p130 negative (0%) N° (%) Rb2/p130 positive (1-30%) N° (%) Rb2/p130 positive(>30%) N° (%) P HISTOLOGIC TYPES Serous Sero-Mucinous subtype Mucinous subtype 26 19 20 26 (100) 17 (89.4) 4 (20.0) 0 1 (5.3) 7 (35.0) 0 1 (5.3) 9 (45.0) <0.0001 Numbers in parentheses represent the percentage of specimens achieving that particular score The cytoplasmic expression of Rb2/p130 was detected in 18 (27.7%) cases whereas it was not evident in the remaining 47 cases (72.3%). Unlike nuclear Rb2/p130 expression, the cytoplasmic expression of Rb2/p130 was not detected in serous tumors, rarely detected in sero-mucinous (2/19, 10.6%) and frequently observed in mucinous subtypes (16/20, 80%) (Fig. 3 ); this difference was statistically significant (P < 0.0001). The cytoplasmic expression of Rb2/p130 according to clinico-pathological parameters is shown in Table 3B . In Fig. 4 we have shown Rb/p130 immunohistochemistry in an intestinal type mucinous ovarian tumor composed of benign, borderline and malignant areas. Notably, the Rb2/p130 expression moves from nuclear expression in the benign counterpart, to nuclear-cytoplasmic in the BOT counterpart and cytoplasmic in the malignant counterpart (Fig. 4 , A/B/C). Discussion Borderline ovarian tumors (BOTs) represent about 15–20% of all ovarian malignancies and differ from invasive ovarian cancers by many characters. On the one hand, they are characterized by cellular proliferation and nuclear atypia but, on the other hand, they usually do not show infiltrative growth pattern. Recent knowledge supports the notion that subtypes of borderline ovarian tumors comprise distinct biologic, pathogenetic, and molecular entities, precluding a single unifying concept for BOT. Serous borderline tumors (SBT) share molecular and genetic alterations with low-grade serous carcinomas and can present at higher stages with peritoneal implants and/or lymph node involvement, which validates their borderline malignant potential. All other (non-serous) subtypes of BOT commonly present at stage I confined to the ovary(ies) and are associated with overall survival approaching that of the general population. The retinoblastoma (Rb) gene family includes Rb2/p130, RB/p105 and p107 genes, which encode nuclear proteins (pRB) acting as negative regulators of cell proliferation, when in their dephosphorylated status. Alteration of Rb family members is frequently involved in gynaecological cancers [ 21 – 23 ]. Dong et al., in a series of 168 specimens, demonstrated high pRb expression in 41% percent of the benign, 50% of the borderline and 71% of the malignant tumors; in this study, protein accumulation increased progressively with poorer differentiation and there was a trend for high pRb expression to be associated with advanced stage of disease [ 25 ]. Additionally, Milde-Langosch et al. demonstrated a correlation between higher pRb expression and shorter survival suggesting thus that pRb expression could play a role in early tumorigenesis, while in later stages, the tumor is independent from pRb [ 26 ]. Other authors reported significantly lower pRb levels in low malignant potential ovarian tumors (LMP) than in carcinomas and in this latter group, a reduction of pRb expression with increasing grade, advancing stage and bulk residual disease; in their study a low pRb to Ki-67 ratio appeared as an indicator of poor survival in uni- and multivariate analysis, along with histologic type and FIGO stage [ 27 ]. We were the first to demonstrate that loss of Rb2/p130 or its cytoplasmic expression occurs in 40% of ovarian tumors and is inversely correlated with tumor grade [ 24 ]. This has been confirmed by Worley MJ et al. who evaluated the immunohistochemical Rb2/p130 expression in a series of benign, borderline (SBT), and malignant ovarian tumors (low-grade (LGSC) and high-grade (HGSC) serous carcinoma), demonstrating a significant decrease in Rb2/p130 expression during the progression from cystadenoma to SBT to LGSC. They reported no loss of expression in benign forms, whereas 10% of SBTs, 47% of LGSCs and 16% of HGSCs had loss of expression [ 28 ]. Differences in additional molecular markers support the idea that the serous BOTs are histologically and clinically distinct from the mucinous BOTs. For example, the expression of p21 and MDM2 differs between mucinous and serous LMP tumors [ 11 ]. In other studies, a higher rate of p53 mutation was observed in mucinous relative to serous BOTs and p21 and bcl-2 overexpression appeared specific to serous BOTs and different among benign, borderline and malignant forms [ 12 , 13 ]. In the present study, the observed differences in the expression of pRb/p105 and pRb2/p130 between serous, sero-mucinous and mucinous BOTs supports the concept that these tumors follow different pathogenic pathways. In our series, the nuclear expression of Rb/p105 and pRb2/p130 was highly detected in serous (84.6%) compare to sero-mucinous (42.1%) and mucinous (50%) types. On the other hand, the cytoplasmic expression of Rb2/p130 was not detected in serous tumors but frequently observed in mucinous subtypes (80%). Our findings suggest that both pRb and pRb2/p130 do not play a key role in the tumor progression of serous borderline tumors since these proteins remain located in the nucleus and never showed a cytoplasmic localization. By contrast, the observed higher cytoplasmic expression of Rb2/p130 in mucinous BOTs, is suggestive of the involvement of Rb proteins in the carcinogenesis of mucinous ovarian tumors. To furtherly support our hypothesis, in Fig. 4 we have shown pRb2/p130 immunohistochemistry in an intestinal type mucinous ovarian tumor composed of benign, borderline and malignant areas. Notably, the Rb2/p130 expression moves from nuclear expression in the benign counterpart, to nuclear-cytoplasmic in the BOT counterpart and cytoplasmic in the malignant counterpart. Despite no statistically significant relationships between pRb immunohistochemistry and prognosis have been observed, our results may suggest that mucinous BOTs, exhibiting low nuclear and high cytoplasmic levels of Rb2/p130, may potentially be considered the BOT histotype with a higher carcinogenic risk. In fact, loss of pRb2/p130 expression has been previously reported to inversely correlate with tumor grade and to be a poor prognostic indicator in several human cancers [ 14 , 14 , 16 , 18 ]. Moreover, its cytoplasmic localization, which implicates a loss of function, has been observed in several tumor types, including lymphoma and gastric cancer [ 29 ]. On the other hand, the normal pRb2/p130 nuclear localization, as more frequently observed in our series for serous and sero-mucinous BOT histotypes, enables its oncosuppressive function through the interaction with the E2F4 and E2F5 transcription factors. Conclusion In conclusion, we have demonstrated a specific histology-related Rb proteins profile of serous, sero-mucinous and mucinous borderline tumors. Our findings indicate a clear role of pRb2/p130 protein in the tumor progression of intestinal-type mucinous BOTs thus suggesting a possible role of Rb proteins as prognostic factors in ovarian cancer. Further studies on larger series are needed in order to clarify how BOTs could be stratified in different prognostic groups according to their pRb immunohistochemical profile. Abbreviations BOT Borderline Ovarian Tumor; RB:Retinoblastoma; pRb:Retinoblastoma protein product or hypophosphorylated form; p-pRb:hyperphosphorylated form. Declarations • Ethics approval and consent to participate: • This study was approved by institutional ethics review board. All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. Informed consent was obtained from the participants included in the study. • Consent for publication: • not applicable • Availability of data and materials: • all the data and materials supporting the conclusions were included in the main paper. • Competing interests: • the authors declare that they have no conflict of interest • Funding: the present research received no fundings • Authors' contributions: Study conceptualization and methodology design: VM and GFZ; Formal analysis and the original draft preparation: GA, AS, GS, PA; Review and editing: GA, As, AG. • Acknowledgements: • not applicable References Seidman JD, Kurman RJ. 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Expression and prognostic value of the cell-cycle regulatory proteins, Rb, p16MTS1, p21WAF1, p27KIP1, cyclin E, and cyclin D2, in ovarian cancer. Int J Gynecol Pathol. 2003;22:168–74. Konstantinidou AE, Korkolopoulou P, Vassilopoulos I, Tsenga A, Thymara I, Agapitos E, et al. Reduced retinoblastoma gene protein to Ki-67 ratio is an adverse prognostic indicator for ovarian adenocarcinoma patients. Gynecol Oncol. 2003;88:369–78. Worley MJ Jr, Landen CN, Slomovitz BM, Malpica A, Palla SL, Ramirez PT. Expression of the retinoblastoma-related gene Rb2/p130 in the pathogenesis of serous carcinoma of the ovary. Appl Immunohistochem Mol Morphol. 2010;18:509–11. Cito L, Pentimalli F, Forte I, Mattioli E, Giordano A. Rb family proteins in gastric cancer (review). Oncol Rep. 2010;24:1411–8. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-48452","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research","associatedPublications":[],"authors":[{"id":1022006,"identity":"a3c77887-83b0-4b26-8d85-d4339d317afd","order_by":0,"name":"Valeria Masciullo","email":"data:image/png;base64,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","orcid":"","institution":"Policlinico Universitario Agostino Gemelli","correspondingAuthor":true,"prefix":"","firstName":"Valeria","middleName":"","lastName":"Masciullo","suffix":""},{"id":1022007,"identity":"05bb13d1-a9d4-4237-ac85-8bdcebe82fcc","order_by":1,"name":"Paola Valdivieso","email":"","orcid":"","institution":"Policlinico Universitario Agostino Gemelli","correspondingAuthor":false,"prefix":"","firstName":"Paola","middleName":"","lastName":"Valdivieso","suffix":""},{"id":1022008,"identity":"017fe5e1-3f4c-4a6c-b066-6520fe3f5382","order_by":2,"name":"Giulia Amadio","email":"","orcid":"","institution":"Policlinico Universitario Agostino Gemelli","correspondingAuthor":false,"prefix":"","firstName":"Giulia","middleName":"","lastName":"Amadio","suffix":""},{"id":1022009,"identity":"eae587ee-c686-400b-b3bd-8dadd3d111a0","order_by":3,"name":"Angela Santoro","email":"","orcid":"","institution":"Policlinico Universitario Agostino Gemelli","correspondingAuthor":false,"prefix":"","firstName":"Angela","middleName":"","lastName":"Santoro","suffix":""},{"id":1022010,"identity":"3b4a4481-749a-4a8d-b6e0-403669d8c2e6","order_by":4,"name":"Giuseppe Angelico","email":"","orcid":"","institution":"Policlinico Universitario Agostino Gemelli","correspondingAuthor":false,"prefix":"","firstName":"Giuseppe","middleName":"","lastName":"Angelico","suffix":""},{"id":1022011,"identity":"8951a2b5-4ad8-4979-9cad-1c8f96da10a3","order_by":5,"name":"Alessandro Sgambato","email":"","orcid":"","institution":"Policlinico Universitario Agostino Gemelli","correspondingAuthor":false,"prefix":"","firstName":"Alessandro","middleName":"","lastName":"Sgambato","suffix":""},{"id":1022012,"identity":"22089952-dfb2-4cee-a39d-81c2f8ca8f6d","order_by":6,"name":"Silvia Boffo","email":"","orcid":"","institution":"Temple University","correspondingAuthor":false,"prefix":"","firstName":"Silvia","middleName":"","lastName":"Boffo","suffix":""},{"id":1022013,"identity":"7ebf6b3b-811c-4fab-9671-7ce029da826b","order_by":7,"name":"Antonio Giordano","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Antonio","middleName":"","lastName":"Giordano","suffix":""},{"id":1022014,"identity":"1ad3dcd7-d271-4cc9-9255-f801a0414f2a","order_by":8,"name":"Giovanni Scambia","email":"","orcid":"","institution":"Policlinico Universitario Agostino Gemelli","correspondingAuthor":false,"prefix":"","firstName":"Giovanni","middleName":"","lastName":"Scambia","suffix":""},{"id":1022015,"identity":"39354967-5c93-4d09-af8f-4bc3c0000baa","order_by":9,"name":"Gian Franco Zannoni","email":"","orcid":"","institution":"Policlinico Universitario Agostino Gemelli","correspondingAuthor":false,"prefix":"","firstName":"Gian","middleName":"Franco","lastName":"Zannoni","suffix":""}],"badges":[],"createdAt":"2020-07-24 10:51:15","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-48452/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-48452/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":1709734,"identity":"18e23797-4b56-467c-9f37-a86c665a97f5","added_by":"auto","created_at":"2020-07-28 17:34:12","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":92942,"visible":true,"origin":"","legend":"Immunohistochemical staining of Rb/p105 expression in the cells nuclei \n(A) Colon Carcinoma as positive control for pRb (X400).\n(B) Brenner BOT as negative control for pRb (X400). \n(C) Mucinous BOT of intestinal type showing a concentration of positive nuclei at the base of the papillar projection (“Arrow”; X200). \n(D) Sero-mucinous BOT with intermediate staining of positive nuclei (X200).\n(E) Follicolar cyst as an example of internal positive control for pRb (X200). \n(F) Serous BOT with lower pRb staining (X200).\n","description":"","filename":"FIGURE1300DPI.jpg","url":"https://assets-eu.researchsquare.com/files/rs-48452/v1/FIGURE1300DPI.jpg"},{"id":1709735,"identity":"cab02767-e0de-4bcb-b6b0-5a761de5d45b","added_by":"auto","created_at":"2020-07-28 17:34:12","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":94168,"visible":true,"origin":"","legend":"Immunohistochemical staining of Rb2/p130 expression \n(A) Tube as internal positive control for Rb2/p130 (X100)\n(B) Mucinous benign tumor with positive nuclei for Rb2/p130 (X200).\n(C) Serous BOT with nuclear Rb2/p130 staining (X200). \n(D) Sero-mucinous BOT with intermediate staining of positive nuclei (X200). \n(E) Mucinous BOT showing cytoplasmic Rb2/p130 staining (X200).\n","description":"","filename":"FIGURE2300DPI.jpg","url":"https://assets-eu.researchsquare.com/files/rs-48452/v1/FIGURE2300DPI.jpg"},{"id":1709736,"identity":"9793efd0-9edf-4d05-a868-6d23ab4e78ba","added_by":"auto","created_at":"2020-07-28 17:34:12","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":24222,"visible":true,"origin":"","legend":"Rb2/p130 cytoplasmic expression in BOTs\nThe graph shows cytoplasmic distribution of Rb2/p130 expression according to histologic types, \nwith focus on the percentage of stained neoplastic cells\n","description":"","filename":"FGURE3300dpi.jpg","url":"https://assets-eu.researchsquare.com/files/rs-48452/v1/FGURE3300dpi.jpg"},{"id":1709737,"identity":"49428a01-fe72-4fcb-817b-d626480226b1","added_by":"auto","created_at":"2020-07-28 17:34:12","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":42986,"visible":true,"origin":"","legend":"Rb/p30 expression in Mucinous BOTs\nA-B) Rb/p30 expression shows a mix of patterns, in a mucinous BOT including combination of benign, borderline and invasive carcinoma. In particular Rb/p130 expression is nuclear in benign area (A, X200) and nuclear-cytoplasmic in borderline component (B, X200); cytoplasmic positivity has been observed in invasive carcinoma (B, X200).\n","description":"","filename":"FIGURE4300DPI.jpg","url":"https://assets-eu.researchsquare.com/files/rs-48452/v1/FIGURE4300DPI.jpg"},{"id":13560636,"identity":"0e1aff6c-21b4-4bad-92a0-aad4d30466e5","added_by":"auto","created_at":"2021-09-17 03:05:36","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":764824,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-48452/v1/d24a41c9-6d4e-4b8f-98aa-8288eeddb65b.pdf"}],"financialInterests":"","formattedTitle":"\u003cp\u003eRole of Retinoblastoma Protein Family (Rb/P105 And Rb2/P130) Expression in the Hystopathological Classification of Borderline Ovarian Tumors \u003c/p\u003e","fulltext":[{"header":"Background","content":" \u003cp\u003eBorderline ovarian tumors (BOTs) represent one of the controversial topics in gynecologic pathology [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. They are a heterogeneous group of tumors that account for 10\u0026ndash;20% of all ovarian epithelial neoplasms. The most common BOT hystotypes are serous (50%) and mucinous (45%) with less common subtypes (5%) including sero-mucinous, endometrioid, clear cell, and borderline Brenner tumors [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. The 97% of all stages of BOT have a good prognosis with a mean 10-years survival [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e], although recurrences and malignant transformation can occur in a very small proportion of cases [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. In contrast to serous BOTs, that are rarely characterized by evolution in a low grade serous carcinoma whereas are often associated to peritoneal implants and relapses [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e], mucinous carcinoma frequently develops from benign and borderline mucinous tumors [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Similarly sero-mucinous BOTs are often the land of endometrioid or clear cell carcinoma and usually represent a morphologic continuum in the middle of benign and malignant counterparts.\u003c/p\u003e \u003cp\u003eBOTs often occur in young women, however the absence of stromal invasion warrant a better prognosis compared to ovarian carcinoma [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Neverthless, since the standard treatment of BOTs is usually surgery, the fertility of these women may be affected [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Identifying the genetic background for diagnosis and prognosis should avoid a radical resection and help in developing new targeted therapies, especially in younger woman with desire of childbearing. Thus, a better understanding of the clinical phenotype and pathogenesis of BOTs would contribute to their earlier detection and is essential for development of more effective treatments.\u003c/p\u003e \u003cp\u003ePrevious studies support the idea that the serous and mucinous BOT have distinct carcinogenic pathways. For example, the expression of p21 and MDM2 differs between mucinous and serous forms [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. In other studies, a higher rate of p53 mutation was observed in mucinous compared to serous BOTs and p21 and bcl-2 overexpression appeared specific to serous forms and different between serous benign, serous BOTs and serous carcinoma [\u003cspan additionalcitationids=\"CR13\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe retinoblastoma gene family consists of three members and their products are: Rb/p105, Rb2/p130 and RbL1/p107, together known as \u0026ldquo;pocket proteins\u0026rdquo; family [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Their most important target is the E2F-family of transcription factors which control the expression of genes that mediate G1-S transition [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. The localization of these proteins into the nucleus or around the nuclear membrane has been shown at the molecular level during the different phases of the cell cycle [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. In detail, during the cell progression through the S into the G2/M phases of division cycle, pRB undergoes phosphorylation, while in the late M phase, pRB is rapidly dephosphorylated. When pRB is in the unphosphorylated form, it acts as a growth suppressor by repressing transcription of E2F. By contrast, the phosphorylated pRB status (p-pRB) leads to activation of E2F-responsive genes and entry into the S phase. While Rb/p105 is ubiquitously confined to the nuclei of cycling and quiescent cells, Rb2/p130 activity is also regulated by intracellular localization. The phosphorylation status of Rb2/p130 itself, therefore, is important in the regulation of cell cycle [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. The hyperphosphorylated form of pRb2/p130 is cytoplasmic and typical of cells progressing into G1 phase [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Alteration of Rb family members is frequently involved in gynaecological cancers [\u003cspan additionalcitationids=\"CR22\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. We previously showed that the loss of Rb2/p130 or its cytoplasmic expression occurs in 40% of ovarian tumors and is inversely correlated with tumor grade [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. However, little is known about Rb proteins expression in borderline ovarian tumors.\u003c/p\u003e \u003cp\u003eIn this study, we utilized immunohistochemistry to evaluate the expression pR/p105 and pRb2/p130 family members in a large, single-institution, series of mucinous, sero-mucinous and serous BOTs.\u003c/p\u003e "},{"header":"Materials And Methods","content":" \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eData collection\u003c/h2\u003e \u003cp\u003eSixty-five ovarian BOTs were retrospectively collected from patients who underwent salpingo-oophorectomy for ovarian cancer in the Division of Gynaecologic Oncology of the Fondazione Policlinico Universitario A.Gemelli IRCSS, Rome, Italy between 2010 and 2016; all selected patients did not receive chemotherapy or radiotherapy before surgical enucleation. All the subjects gave a written informed consent before enrollment. Twenty specimens were defined as mucinous BOTs, 19 were classified as sero-mucinous, whereas 26 cases were classified as serous BOTs. Histological classification of tumors was carried out according to the WHO system, and disease staging was established according to the International Federation of Gynaecologist and Obstetricians (FIGO) criteria.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eImmunohistochemistry\u003c/h2\u003e \u003cp\u003eAfter surgical resection, tissues were immediately fixed in 10% formalin and then paraffin-embedded for immunohistochemical analysis. The immunostaining was performed using a streptavidin-biotin complex immuno peroxidase method (DakoCytomation). Detection for the retinoblastoma gene family members was performed using purified mouse anti-human retinoblastoma protein (Rb) monoclonal antibody (BD Pharmigen) diluted 1:50 and mouse monoclonal antibody Rb2 p130 (clone 130-P215; Novus Biologicals, Inc.) diluted 1:25.\u003c/p\u003e \u003cp\u003eParaffin blocks of each specimen were sectioned at 3\u0026nbsp;\u0026micro;m, mounted on slide and dried overnight at 37\u0026nbsp;\u0026deg;C. All sections were dewaxed in xylene and dehydrated in descending graded alcohols to Phosphate-Buffered Saline (PBS; pH 7.4).\u003c/p\u003e \u003cp\u003eAntigen retrieval was performed by microwaves in 10\u0026nbsp;mM Citrate buffer (pH 6), at 750\u0026nbsp;W for 10\u0026nbsp;min (two cycles of 5\u0026nbsp;min each), followed by cooling at room temperature for at least 20\u0026nbsp;min prior to incubation with the antibodies. Sections were treated with 0.4% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e methanol solution (15\u0026nbsp;min at room temperature to inhibit endogenous peroxidase activity), quickly rinsed in water and then in PBS.\u003c/p\u003e \u003cp\u003eSections were then placed in a humidified chamber and incubated with primary antibody at room temperature for 40\u0026nbsp;min. The sections were then washed in PBS (two times for 5\u0026nbsp;min each).\u003c/p\u003e \u003cp\u003eAntigen detection was carried out by exposure to a biotinylated universal secondary antibody for 10\u0026nbsp;min followed by a streptavidin-peroxidase complex working solution for 10\u0026nbsp;min.\u003c/p\u003e \u003cp\u003eAfter another PBS wash, the antigen antibody complex was visualized by staining with chromogen 3,3\u0026rsquo;-diaminobenzidine/ tetrachloride solution (DAB, Vector) for 5\u0026nbsp;min. The sections were rinsed in deionized water; cell nuclei were counterstained with hematoxylin and dehydrated in graded alcohols followed by xylene.\u003c/p\u003e \u003cp\u003eSpecimens of human colon cancer, follicolar cyst and falloppian tube served as positive controls for pRb and pRb2, respectively. For negative control, slides were simultaneously incubated with PBS in the absence of the primary antibody. The results were independently reviewed by three experienced pathologists (GFZ, GA, AS), who were blinded to clinical outcome at the time of evaluation. Discrepancies in the evaluation (\u0026lt;\u0026thinsp;10% of cases) were resolved by re-observation of the cases using a multi-headed microscope.\u003c/p\u003e \u003cp\u003eQuantitative scoring of protein expression was based on the staining intensity and the percentage of positive cells as follows: negative (0%); lower positive (1\u0026ndash;30%) or upper positive (\u0026gt;\u0026thinsp;30%) cells. A cut-off of 30% was considered statistically significant and, therefore, functionally operative.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eThe associations between Rb proteins staining and other clinic-pathological parameters were analysed using contingency table methods and tested for significance using the Fisher\u0026rsquo;s exact χ2 test. All calculation were performed using the Statistical Package for Social Science (SPSS 17.0 software, Chicago, IL) and the result was considered statistically significant when the P value was less or equal to 0.05.\u003c/p\u003e \u003c/div\u003e "},{"header":"Results","content":" \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eClinico-pathological features\u003c/h2\u003e \u003cp\u003eThe series included 20 mucinous, 19 sero-mucinous and 26 serous BOTs. The mean and median age of the patients were 44.7 and 44\u0026nbsp;years (range 20\u0026ndash;72), respectively. All mucinous and sero-mucinous selected tumors (39) were Stage 1, without evidence of implants or recurrences, whereas serous tumors included stage 1 (n\u0026thinsp;=\u0026thinsp;20), stage 2 (n\u0026thinsp;=\u0026thinsp;1) and stage 3 (n\u0026thinsp;=\u0026thinsp;5) cases and follow-up data were available for all 65 patients. Five serous BOTs were classified as BOTs with microinvasive foci. One mucinous BOT showed foci of intraepithelial carcinoma and an area of malignant invasive mucinous carcinoma with expansive pattern of growth.\u003c/p\u003e \u003cp\u003eFifty-nine cases were limited to the ovary, without peritoneal implants and 6 cases (all serous BOTs) were associated to peritoneal implants (only 1 of invasive type). Sixty-one cases did not relapse; the remaining 4 developed peritoneal recurrences. By the time this study was undertaken, no patients had died of the disease.\u003c/p\u003e \u003cp\u003eClinico-pathologic characteristics of patients are summarized in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The expression levels of Rb/p105 and Rb2/p130 were determined by immunohistochemistry.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eClinical data of the patients with BOTs\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"2\"\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHISTOLOGIC TYPES\u003c/p\u003e \u003cp\u003eSerous subtype*\u003c/p\u003e \u003cp\u003eSero-Mucinous subtype\u003c/p\u003e \u003cp\u003eMucinous subtype**\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eN\u0026deg; (% of Cases)\u003c/p\u003e \u003cp\u003e26 (40.0)\u003c/p\u003e \u003cp\u003e19 (29.2)\u003c/p\u003e \u003cp\u003e20 (30.8)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eSTAGE\u003c/b\u003e\u003c/p\u003e \u003cp\u003e1\u003c/p\u003e \u003cp\u003e2\u003c/p\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eN\u0026deg; (% of Cases)\u003c/b\u003e\u003c/p\u003e \u003cp\u003e59 (90.8)\u003c/p\u003e \u003cp\u003e1 (1.5)\u003c/p\u003e \u003cp\u003e5 (7.7)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eIMPLANTS\u003c/b\u003e\u003c/p\u003e \u003cp\u003eNot Implants\u003c/p\u003e \u003cp\u003eImplants without invasion\u003c/p\u003e \u003cp\u003eImplants with invasion\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eN\u0026deg; (% of Cases)\u003c/b\u003e\u003c/p\u003e \u003cp\u003e59 (90.8)\u003c/p\u003e \u003cp\u003e5 (7.7)\u003c/p\u003e \u003cp\u003e1 (1.5)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eRECURRENCES\u003c/b\u003e\u003c/p\u003e \u003cp\u003eNo\u003c/p\u003e \u003cp\u003eYes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eN\u0026deg; (% of Cases)\u003c/b\u003e\u003c/p\u003e \u003cp\u003e61 (93.8)\u003c/p\u003e \u003cp\u003e4 (6.2)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eCLINICAL OUTCOME\u003c/b\u003e\u003c/p\u003e \u003cp\u003eDead\u003c/p\u003e \u003cp\u003eAlive\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eN\u0026deg; (% of Cases)\u003c/b\u003e\u003c/p\u003e \u003cp\u003e0\u003c/p\u003e \u003cp\u003e65 (100)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"2\"\u003e\u003cem\u003e* 5 serous BOTs showed micro-invasive foci\u003c/em\u003e\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"2\"\u003e\u003cem\u003e** 1 mucinous BOT showed foci of intraepithelial carcinoma and an area of invasive mucinous carcinoma with expansive pattern of growth\u003c/em\u003e\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eCorrelation of Rb/p105 with clinic-pathological parameters in BOTs.\u003c/span\u003e \u003c/p\u003e \u003cp\u003eThe expression of Rb/p105 according to clinico-pathological parameters is shown in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. Notably, the immunoreactivity for Rb/p105 was only nuclear in our series. Moreover, the nuclear Rb staining in intestinal type mucinous BOTs tended to concentrate at the bases of the papillary projections (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eNuclear Distribution of Rb/p105-negative and Rb/p105-positive cases according to tumoral characteristics\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRb/p105 Nuclear\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTotal\u003c/p\u003e \u003cp\u003eN\u0026deg;\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eRb/p105 negative (0%)\u003c/p\u003e \u003cp\u003eN\u0026deg; (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eRb/p105 positive (1\u0026ndash;30%)\u003c/p\u003e \u003cp\u003eN\u0026deg; (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eRb/p105 positive (\u0026gt;\u0026thinsp;30%)\u003c/p\u003e \u003cp\u003eN\u0026deg; (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eP\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eHISTOLOGIC TYPES\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e65\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e25 (38.4)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e33 (50.8)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e7 (10.8)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSerous\u003c/p\u003e \u003cp\u003eSero-Mucinous subtype\u003c/p\u003e \u003cp\u003eMucinous subtype\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e26\u003c/p\u003e \u003cp\u003e19\u003c/p\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4\u003c/p\u003e \u003cp\u003e11 (57.9)\u003c/p\u003e \u003cp\u003e10 (50.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e22 (84.6)\u003c/p\u003e \u003cp\u003e6 (31.6)\u003c/p\u003e \u003cp\u003e5 (25.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0 (15.4.)\u003c/p\u003e \u003cp\u003e2 (10.5)\u003c/p\u003e \u003cp\u003e5 (25.0))\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eSTAGE\u003c/b\u003e\u003c/p\u003e \u003cp\u003e1\u003c/p\u003e \u003cp\u003e2\u0026thinsp;+\u0026thinsp;3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e59\u003c/p\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e25 (42.3)\u003c/p\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e27 (45.8)\u003c/p\u003e \u003cp\u003e6 (100)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e7 (11.9)\u003c/p\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.04\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eIMPLANTS\u003c/b\u003e\u003c/p\u003e \u003cp\u003eAbsent\u003c/p\u003e \u003cp\u003ePresent\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e59\u003c/p\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e24 (41.4)\u003c/p\u003e \u003cp\u003e1 (14.3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e27 (46.5)\u003c/p\u003e \u003cp\u003e6 (85.7)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e7 (12.1)\u003c/p\u003e \u003cp\u003e(0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNS\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eRECURRENCES\u003c/b\u003e\u003c/p\u003e \u003cp\u003eAbsent\u003c/p\u003e \u003cp\u003ePresent\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e61\u003c/p\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e24 (39.4)\u003c/p\u003e \u003cp\u003e1 (25.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e31 (50.8)\u003c/p\u003e \u003cp\u003e2 (50.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e6 (9.8)\u003c/p\u003e \u003cp\u003e1 (25.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNS\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"6\"\u003e\u003cem\u003eNumbers in parentheses represent the percentage of specimens achieving that particular score. NS, not significant.\u003c/em\u003e\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe nuclear expression of Rb/p105 was observed in 40 (61.6%) out of the 65 patients, whereas it was not detectable in the remaining 25 cases (38.4%). The expression of nuclear Rb/p105 was more frequently detected in serous (22 cases; 84.6%) than in serous mucinous (8/19, 42.1%) and mucinous (10/20, 50%) types, and this difference was statistically significant (P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). Positive staining was observed in 34 (57.6%) out of 59 stage 1 and in all 6 (100%) stage 2/3 cases and this difference was slightly significant (p\u0026thinsp;=\u0026thinsp;0.04). No statistically significant correlation was observed between the nuclear expression of Rb/p105, implants and recurrences (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eCorrelation of Rb2/p130 expression with clinico-pathological parameters in BOTs\u003c/h2\u003e \u003cp\u003eNuclear Rb2/p130 expression was detected in 33 (50.8%) of the 65 cases whereas it was absent in the remaining 32 cases (40.2%) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The expression of nuclear Rb2/p130 was more frequent in serous (21/26, 80.8%) than in sero-mucinous (10/19, 52.6%) and intestinal (2/20, 1.0%) types, and these differences were statistically significant (P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). Positive staining was observed in 27 (45.7%) out of the 59 stage 1 and in all 6 (100%) stage 2/3 cases and this difference was significant (p\u0026thinsp;=\u0026thinsp;0.03). No statistically significant correlation was observed between the nuclear expression of Rb2/p30, implants and recurrences (\u003cb\u003eTable\u0026nbsp;3A\u003c/b\u003e)\u003c/p\u003e \u003cp\u003e\u003cstrong\u003eTable 3 (A)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNuclear d\u003c/strong\u003e\u003cstrong\u003eistribution of Rb2/p130-negative and Rb2/p130-positive cases according to tumoral characteristics\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"1\" width=\"685\"\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd width=\"171\"\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eRb2/p130 Nuclear\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"37\"\u003e\n\u003cp\u003e\u003cstrong\u003eTotal\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eN\u0026deg;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"137\"\u003e\n\u003cp\u003e\u003cstrong\u003eRb2/p130 negative (0%)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eN\u0026deg; (%)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"151\"\u003e\n\u003cp\u003e\u003cstrong\u003eRb2/p130 positive (1-30%)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eN\u0026deg; (%)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"141\"\u003e\n\u003cp\u003e\u003cstrong\u003eRb2/p130 positive(\u0026gt;30%)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eN\u0026deg; (%)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"48\"\u003e\n\u003cp\u003e\u003cstrong\u003eP\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"171\"\u003e\n\u003cp\u003e\u003cstrong\u003eHISTOLOGIC TYPES\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSerous\u003c/p\u003e\n\u003cp\u003eSero-Mucinous subtype\u003c/p\u003e\n\u003cp\u003eMucinous subtype\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"37\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e26\u003c/p\u003e\n\u003cp\u003e19\u003c/p\u003e\n\u003cp\u003e20\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"137\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e5 (19.2)\u003c/p\u003e\n\u003cp\u003e9 (47.4)\u003c/p\u003e\n\u003cp\u003e18 (90.0)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"151\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e7 (26.9)\u003c/p\u003e\n\u003cp\u003e5 (26.3)\u003c/p\u003e\n\u003cp\u003e2(10.0)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"141\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e14 (53.9)\u003c/p\u003e\n\u003cp\u003e5 (26.3)\u003c/p\u003e\n\u003cp\u003e0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"48\"\u003e\n\u003cp\u003e\u0026lt;0.0001\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"171\"\u003e\n\u003cp\u003eSTAGE\u003c/p\u003e\n\u003cp\u003e1\u003c/p\u003e\n\u003cp\u003e2+3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"37\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e59\u003c/p\u003e\n\u003cp\u003e6\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"137\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e32 (54.3)\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"151\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e12 (20.3)\u003c/p\u003e\n\u003cp\u003e2 (33.3)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"141\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e15 (25.4)\u003c/p\u003e\n\u003cp\u003e4 (66.7)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"48\"\u003e\n\u003cp\u003e0.03\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"171\"\u003e\n\u003cp\u003eIMPLANTS\u003c/p\u003e\n\u003cp\u003eAbsent\u003c/p\u003e\n\u003cp\u003ePresent\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"37\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e59\u003c/p\u003e\n\u003cp\u003e6\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"137\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e31 (53.4)\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;1\u0026nbsp; (14.3)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"151\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e12 (20.7)\u003c/p\u003e\n\u003cp\u003e2 (28.6)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"141\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e15 (25.9)\u003c/p\u003e\n\u003cp\u003e4 (57.1)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"48\"\u003e\n\u003cp\u003eNS\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"171\"\u003e\n\u003cp\u003eRECURRENCES\u003c/p\u003e\n\u003cp\u003eAbsent\u003c/p\u003e\n\u003cp\u003ePresent\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"37\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e61\u003c/p\u003e\n\u003cp\u003e4\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"137\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e30 (49.2)\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; 2 (50.0)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"151\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e12 (19.7)\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; 2 (50.0)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"141\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e19 (31.1)\u003c/p\u003e\n\u003cp\u003e0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"48\"\u003e\n\u003cp\u003eNS\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cem\u003eNumbers in parentheses represent the percentage of specimens achieving that particular score. \u003c/em\u003e\u003cem\u003eNS, not significant\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 3 (B)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCytoplasmic distribution of Rb2/p130-negative and Rb2/p130-positive cases according to histologic types\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"1\" width=\"685\"\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd width=\"173\"\u003e\n\u003cp\u003e\u003cstrong\u003eRb2/p130 Cytoplasmatic\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"27\"\u003e\n\u003cp\u003e\u003cstrong\u003eTotal\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eN\u0026deg;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"139\"\u003e\n\u003cp\u003e\u003cstrong\u003eRb2/p130 negative (0%)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eN\u0026deg; (%)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"153\"\u003e\n\u003cp\u003e\u003cstrong\u003eRb2/p130 positive (1-30%)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eN\u0026deg; (%)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"143\"\u003e\n\u003cp\u003e\u003cstrong\u003eRb2/p130 positive(\u0026gt;30%)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eN\u0026deg; (%)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"48\"\u003e\n\u003cp\u003e\u003cstrong\u003eP\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"173\"\u003e\n\u003cp\u003e\u003cstrong\u003eHISTOLOGIC TYPES\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSerous\u003c/p\u003e\n\u003cp\u003eSero-Mucinous subtype\u003c/p\u003e\n\u003cp\u003eMucinous subtype\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"27\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e26\u003c/p\u003e\n\u003cp\u003e19\u003c/p\u003e\n\u003cp\u003e20\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"139\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e26 (100)\u003c/p\u003e\n\u003cp\u003e17 (89.4)\u003c/p\u003e\n\u003cp\u003e4 (20.0)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"153\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e0\u003c/p\u003e\n\u003cp\u003e1 (5.3)\u003c/p\u003e\n\u003cp\u003e7 (35.0)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"143\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e0\u003c/p\u003e\n\u003cp\u003e1 (5.3)\u003c/p\u003e\n\u003cp\u003e9 (45.0)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"48\"\u003e\n\u003cp\u003e\u0026lt;0.0001\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cem\u003eNumbers in parentheses represent the percentage of specimens achieving that particular score\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e \u003cp\u003eThe cytoplasmic expression of Rb2/p130 was detected in 18 (27.7%) cases whereas it was not evident in the remaining 47 cases (72.3%). Unlike nuclear Rb2/p130 expression, the cytoplasmic expression of Rb2/p130 was not detected in serous tumors, rarely detected in sero-mucinous (2/19, 10.6%) and frequently observed in mucinous subtypes (16/20, 80%) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e); this difference was statistically significant (P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). The cytoplasmic expression of Rb2/p130 according to clinico-pathological parameters is shown in \u003cb\u003eTable\u0026nbsp;3B\u003c/b\u003e. In Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e we have shown Rb/p130 immunohistochemistry in an intestinal type mucinous ovarian tumor composed of benign, borderline and malignant areas. Notably, the Rb2/p130 expression moves from nuclear expression in the benign counterpart, to nuclear-cytoplasmic in the BOT counterpart and cytoplasmic in the malignant counterpart (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, A/B/C).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e "},{"header":"Discussion","content":" \u003cp\u003eBorderline ovarian tumors (BOTs) represent about 15\u0026ndash;20% of all ovarian malignancies and differ from invasive ovarian cancers by many characters. On the one hand, they are characterized by cellular proliferation and nuclear atypia but, on the other hand, they usually do not show infiltrative growth pattern. Recent knowledge supports the notion that subtypes of borderline ovarian tumors comprise distinct biologic, pathogenetic, and molecular entities, precluding a single unifying concept for BOT. Serous borderline tumors (SBT) share molecular and genetic alterations with low-grade serous carcinomas and can present at higher stages with peritoneal implants and/or lymph node involvement, which validates their borderline malignant potential. All other (non-serous) subtypes of BOT commonly present at stage I confined to the ovary(ies) and are associated with overall survival approaching that of the general population.\u003c/p\u003e \u003cp\u003eThe retinoblastoma (Rb) gene family includes Rb2/p130, RB/p105 and p107 genes, which encode nuclear proteins (pRB) acting as negative regulators of cell proliferation, when in their dephosphorylated status.\u003c/p\u003e \u003cp\u003eAlteration of Rb family members is frequently involved in gynaecological cancers [\u003cspan additionalcitationids=\"CR22\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eDong et al., in a series of 168 specimens, demonstrated high pRb expression in 41% percent of the benign, 50% of the borderline and 71% of the malignant tumors; in this study, protein accumulation increased progressively with poorer differentiation and there was a trend for high pRb expression to be associated with advanced stage of disease [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Additionally, Milde-Langosch et al. demonstrated a correlation between higher pRb expression and shorter survival suggesting thus that pRb expression could play a role in early tumorigenesis, while in later stages, the tumor is independent from pRb [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eOther authors reported significantly lower pRb levels in low malignant potential ovarian tumors (LMP) than in carcinomas and in this latter group, a reduction of pRb expression with increasing grade, advancing stage and bulk residual disease; in their study a low pRb to Ki-67 ratio appeared as an indicator of poor survival in uni- and multivariate analysis, along with histologic type and FIGO stage [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eWe were the first to demonstrate that loss of Rb2/p130 or its cytoplasmic expression occurs in 40% of ovarian tumors and is inversely correlated with tumor grade [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. This has been confirmed by Worley MJ et al. who evaluated the immunohistochemical Rb2/p130 expression in a series of benign, borderline (SBT), and malignant ovarian tumors (low-grade (LGSC) and high-grade (HGSC) serous carcinoma), demonstrating a significant decrease in Rb2/p130 expression during the progression from cystadenoma to SBT to LGSC. They reported no loss of expression in benign forms, whereas 10% of SBTs, 47% of LGSCs and 16% of HGSCs had loss of expression [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eDifferences in additional molecular markers support the idea that the serous BOTs are histologically and clinically distinct from the mucinous BOTs. For example, the expression of p21 and MDM2 differs between mucinous and serous LMP tumors [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. In other studies, a higher rate of p53 mutation was observed in mucinous relative to serous BOTs and p21 and bcl-2 overexpression appeared specific to serous BOTs and different among benign, borderline and malignant forms [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn the present study, the observed differences in the expression of pRb/p105 and pRb2/p130 between serous, sero-mucinous and mucinous BOTs supports the concept that these tumors follow different pathogenic pathways. In our series, the nuclear expression of Rb/p105 and pRb2/p130 was highly detected in serous (84.6%) compare to sero-mucinous (42.1%) and mucinous (50%) types. On the other hand, the cytoplasmic expression of Rb2/p130 was not detected in serous tumors but frequently observed in mucinous subtypes (80%). Our findings suggest that both pRb and pRb2/p130 do not play a key role in the tumor progression of serous borderline tumors since these proteins remain located in the nucleus and never showed a cytoplasmic localization.\u003c/p\u003e \u003cp\u003eBy contrast, the observed higher cytoplasmic expression of Rb2/p130 in mucinous BOTs, is suggestive of the involvement of Rb proteins in the carcinogenesis of mucinous ovarian tumors. To furtherly support our hypothesis, in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e we have shown pRb2/p130 immunohistochemistry in an intestinal type mucinous ovarian tumor composed of benign, borderline and malignant areas. Notably, the Rb2/p130 expression moves from nuclear expression in the benign counterpart, to nuclear-cytoplasmic in the BOT counterpart and cytoplasmic in the malignant counterpart.\u003c/p\u003e \u003cp\u003eDespite no statistically significant relationships between pRb immunohistochemistry and prognosis have been observed, our results may suggest that mucinous BOTs, exhibiting low nuclear and high cytoplasmic levels of Rb2/p130, may potentially be considered the BOT histotype with a higher carcinogenic risk. In fact, loss of pRb2/p130 expression has been previously reported to inversely correlate with tumor grade and to be a poor prognostic indicator in several human cancers [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Moreover, its cytoplasmic localization, which implicates a loss of function, has been observed in several tumor types, including lymphoma and gastric cancer [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eOn the other hand, the normal pRb2/p130 nuclear localization, as more frequently observed in our series for serous and sero-mucinous BOT histotypes, enables its oncosuppressive function through the interaction with the E2F4 and E2F5 transcription factors.\u003c/p\u003e "},{"header":"Conclusion","content":" \u003cp\u003eIn conclusion, we have demonstrated a specific histology-related Rb proteins profile of serous, sero-mucinous and mucinous borderline tumors.\u003c/p\u003e \u003cp\u003eOur findings indicate a clear role of pRb2/p130 protein in the tumor progression of intestinal-type mucinous BOTs thus suggesting a possible role of Rb proteins as prognostic factors in ovarian cancer.\u003c/p\u003e \u003cp\u003eFurther studies on larger series are needed in order to clarify how BOTs could be stratified in different prognostic groups according to their pRb immunohistochemical profile.\u003c/p\u003e "},{"header":"Abbreviations","content":" \u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eBOT\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eBorderline Ovarian Tumor; RB:Retinoblastoma; pRb:Retinoblastoma protein product or hypophosphorylated form; p-pRb:hyperphosphorylated form.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e "},{"header":"Declarations","content":" \u003cp\u003e \u003ch2\u003e\u0026bull; Ethics approval and consent to participate:\u003c/h2\u003e \u003cp\u003e\u0026bull; This study was approved by institutional ethics review board. All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. Informed consent was obtained from the participants included in the study.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003e\u0026bull; Consent for publication:\u003c/strong\u003e \u003cp\u003e\u0026bull; not applicable\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003e\u0026bull; Availability of data and materials:\u003c/strong\u003e \u003cp\u003e\u0026bull; all the data and materials supporting the conclusions were included in the main paper.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003e\u0026bull; Competing interests:\u003c/strong\u003e \u003cp\u003e\u0026bull; the authors declare that they have no conflict of interest\u003c/p\u003e \u003c/p\u003e \u003ch2\u003e\u0026bull; Funding:\u003c/h2\u003e \u003cp\u003ethe present research received no fundings\u003c/p\u003e \u003ch2\u003e\u0026bull; Authors' contributions:\u003c/h2\u003e \u003cp\u003eStudy conceptualization and methodology design: VM and GFZ; Formal analysis and the original draft preparation: GA, AS, GS, PA; Review and editing: GA, As, AG.\u003c/p\u003e \u003ch2\u003e\u0026bull; Acknowledgements:\u003c/h2\u003e \u003cp\u003e\u0026bull; not applicable\u003c/p\u003e "},{"header":"References","content":"\u003col\u003e\u003cli\u003e \u003cspan\u003eSeidman JD, Kurman RJ. Ovarian serous borderline tumors: a critical review of the literature with emphasis on prognostic indicators. Hum Pathol. 2000;31:539\u0026ndash;57.\u003c/span\u003e \u003c/li\u003e \u003cli\u003e \u003cspan\u003eHauptmann S, Friedrich K, Redline R, Avril S. Ovarian borderline tumors in the 2014 WHO classification: evolving concepts and diagnostic criteria. Virchows Arch. 2017;470:125\u0026ndash;42.\u003c/span\u003e \u003c/li\u003e \u003cli\u003e \u003cspan\u003eSiriaunkgul S, Robbins KM, McGowan L, Silverberg SG. Ovarian mucinous tumors of low malignant potential: a clinicopathologic study of 54 tumors of intestinal and mullerian type. Int J Gynecol Pathol. 1995;14:198\u0026ndash;208.\u003c/span\u003e \u003c/li\u003e \u003cli\u003e \u003cspan\u003eSherman ME, Mink PJ, Curtis R, Cote TR, Brooks S, Hartge P, et al. Survival among women with borderline ovarian tumors and ovarian carcinoma: a population-based analysis. Cancer. 2004;100:1045\u0026ndash;52.\u003c/span\u003e \u003c/li\u003e \u003cli\u003e \u003cspan\u003eKurman RJ, Trimble CL. The behavior of serous tumors of low malignant potential: are they ever malignant? Int J Gynecol Pathol. 1993;12:120\u0026ndash;7.\u003c/span\u003e \u003c/li\u003e \u003cli\u003e \u003cspan\u003eLee KR, Scully RE. Mucinous tumors of the ovary: a clinicopathologic study of 196 borderline tumors (of intestinal type) and carcinomas, including an evaluation of 11 cases with 'pseudomyxoma peritonei'. Am J Surg Pathol. 2000;24:1447\u0026ndash;64.\u003c/span\u003e \u003c/li\u003e \u003cli\u003e \u003cspan\u003eRiopel MA, Ronnett BM, Kurman RJ. Evaluation of diagnostic criteria and behavior of ovarian intestinal-type mucinous tumors: atypical proliferative (borderline) tumors and intraepithelial, microinvasive, invasive, and metastatic carcinomas. Am J Surg Pathol. 1999;23:617\u0026ndash;35.\u003c/span\u003e \u003c/li\u003e \u003cli\u003e \u003cspan\u003eHart WR. Borderline epithelial tumors of the ovary. Mod Pathol. 2005;18(Suppl 2):33\u0026ndash;50.\u003c/span\u003e \u003c/li\u003e \u003cli\u003e \u003cspan\u003ePrat J. Pathology of borderline and invasive cancers. Best Pract Res Clin Obstet Gynaecol. 2017;41:15\u0026ndash;30.\u003c/span\u003e \u003c/li\u003e \u003cli\u003e \u003cspan\u003edu Bois A, Trillsch F, Mahner S, Heitz F, Harter P. Management of borderline ovarian tumors. Ann Oncol. 2016;27(Suppl 1):i20-i2.\u003c/span\u003e \u003c/li\u003e \u003cli\u003e \u003cspan\u003ePalazzo JP, Monzon F, Burke M, Hyslop T, Dunton C, Barusevicius A, et al. Overexpression of p21WAF1/CIP1 and MDM2 characterizes serous borderline ovarian tumors. Hum Pathol. 2000;31:698\u0026ndash;704.\u003c/span\u003e \u003c/li\u003e \u003cli\u003e \u003cspan\u003eLee JH, Kang YS, Park SY, Kim BG, Lee ED, Lee KH, et al. p53 mutation in epithelial ovarian carcinoma and borderline ovarian tumor. Cancer Genet Cytogenet. 1995;85:43\u0026ndash;50.\u003c/span\u003e \u003c/li\u003e \u003cli\u003e \u003cspan\u003eFauvet R, Dufournet C, Poncelet C, Uzan C, Hugol D, Darai E. Expression of pro-apoptotic (p53, p21, bax, bak and fas) and anti-apoptotic (bcl-2 and bcl-x) proteins in serous versus mucinous borderline ovarian tumours. J Surg Oncol. 2005;92:337\u0026ndash;43.\u003c/span\u003e \u003c/li\u003e \u003cli\u003e \u003cspan\u003eDong Y, Walsh MD, McGuckin MA, Cummings MC, Gabrielli BG, Wright GR, et al. Reduced expression of retinoblastoma gene product (pRB) and high expression of p53 are associated with poor prognosis in ovarian cancer. Int J Cancer. 1997;74:407\u0026ndash;15.\u003c/span\u003e \u003c/li\u003e \u003cli\u003e \u003cspan\u003eClaudio PP, Howard CM, Baldi A, De Luca A, Fu Y, Condorelli G, et al. p130/pRb2 has growth suppressive properties similar to yet distinctive from those of retinoblastoma family members pRb and p107. Cancer Res. 1994;54:5556\u0026ndash;60.\u003c/span\u003e \u003c/li\u003e \u003cli\u003e \u003cspan\u003ePaggi MG, Baldi A, Bonetto F, Giordano A. Retinoblastoma protein family in cell cycle and cancer: a review. J Cell Biochem. 1996;62:418\u0026ndash;30.\u003c/span\u003e \u003c/li\u003e \u003cli\u003e \u003cspan\u003eClaudio PP, De Luca A, Howard CM, Baldi A, Firpo EJ, Koff A, et al. Functional analysis of pRb2/p130 interaction with cyclins. Cancer Res. 1996;56:2003\u0026ndash;8.\u003c/span\u003e \u003c/li\u003e \u003cli\u003e \u003cspan\u003eMileo AM, Mattarocci S, Matarrese P, et al. Hepatitis C virus core protein modulates pRb2/p130 expression in human hepatocellular carcinoma cell lines through promoter methylation. J Exp Clin Cancer Res. 2015;34:140.\u003c/span\u003e \u003c/li\u003e \u003cli\u003e \u003cspan\u003eCicchillitti L, Fasanaro P, Biglioli P, Capogrossi MC, Martelli F. Oxidative stress induces protein phosphatase 2A-dependent dephosphorylation of the pocket proteins pRb, p107, and p130. J Biol Chem. 2003;278:19509\u0026ndash;17.\u003c/span\u003e \u003c/li\u003e \u003cli\u003e \u003cspan\u003eVerona R, Moberg K, Estes S, Starz M, Vernon JP, Lees JA. E2F activity is regulated by cell cycle-dependent changes in subcellular localization. Mol Cell Biol. 1997;17:7268\u0026ndash;82.\u003c/span\u003e \u003c/li\u003e \u003cli\u003e \u003cspan\u003eSanseverino F, Torricelli M, Petraglia F, Giordano A. Role of the retinoblastoma family in gynecological cancer. Cancer Biol Ther. 2003;2:636\u0026ndash;41.\u003c/span\u003e \u003c/li\u003e \u003cli\u003e \u003cspan\u003eSusini T, Massi D, Paglierani M, et al. Expression of the retinoblastoma-related gene Rb2/p130 is downregulated in atypical endometrial hyperplasia and adenocarcinoma. Hum Pathol. 2001;32:360\u0026ndash;7.\u003c/span\u003e \u003c/li\u003e \u003cli\u003e \u003cspan\u003eZamparelli A, Masciullo V, Bovicelli A, et al. Expression of cell-cycle-associated proteins pRB2/p130 and p27kip in vulvar squamous cell carcinomas. Hum Pathol. 2001;32:4\u0026ndash;9.\u003c/span\u003e \u003c/li\u003e \u003cli\u003e \u003cspan\u003eD'Andrilli G, Masciullo V, Bagella L, Tonini T, Minimo C, Zannoni GF, et al. Frequent loss of pRb2/p130 in human ovarian carcinoma. Clin Cancer Res. 2004;10:3098\u0026ndash;103.\u003c/span\u003e \u003c/li\u003e \u003cli\u003e \u003cspan\u003eDong Y, Walsh MD, McGuckin MA, Cummings MC, Gabrielli BG, Wright GR, et al. Reduced expression of retinoblastoma gene product (pRB) and high expression of p53 are associated with poor prognosis in ovarian cancer. Int J Cancer. 1997;74:407\u0026ndash;15.\u003c/span\u003e \u003c/li\u003e \u003cli\u003e \u003cspan\u003eMilde-Langosch K, Hagen M, Bamberger AM, L\u0026ouml;ning T. Expression and prognostic value of the cell-cycle regulatory proteins, Rb, p16MTS1, p21WAF1, p27KIP1, cyclin E, and cyclin D2, in ovarian cancer. Int J Gynecol Pathol. 2003;22:168\u0026ndash;74.\u003c/span\u003e \u003c/li\u003e \u003cli\u003e \u003cspan\u003eKonstantinidou AE, Korkolopoulou P, Vassilopoulos I, Tsenga A, Thymara I, Agapitos E, et al. Reduced retinoblastoma gene protein to Ki-67 ratio is an adverse prognostic indicator for ovarian adenocarcinoma patients. Gynecol Oncol. 2003;88:369\u0026ndash;78.\u003c/span\u003e \u003c/li\u003e \u003cli\u003e \u003cspan\u003eWorley MJ Jr, Landen CN, Slomovitz BM, Malpica A, Palla SL, Ramirez PT. Expression of the retinoblastoma-related gene Rb2/p130 in the pathogenesis of serous carcinoma of the ovary. Appl Immunohistochem Mol Morphol. 2010;18:509\u0026ndash;11.\u003c/span\u003e \u003c/li\u003e \u003cli\u003e \u003cspan\u003eCito L, Pentimalli F, Forte I, Mattioli E, Giordano A. Rb family proteins in gastric cancer (review). Oncol Rep. 2010;24:1411\u0026ndash;8.\u003c/span\u003e \u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Retinoblastoma family proteins, borderline ovarian tumors, pRb/p105, pRb2/p130, diagnosis","lastPublishedDoi":"10.21203/rs.3.rs-48452/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-48452/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eBorderline ovarian tumors (BOT) are uncommon but not rare epithelial ovarian neoplasms, intermediate between benign and malignant categories. Emerging knowledge supports the notion that subtypes of borderline ovarian tumors comprise distinct biologic, pathogenetic, and molecular entities, precluding a single unifying concept for BOT. Therefore, the identification of valuable markers for the diagnosis and classification of these tumors is in need. Among the molecular candidates, the Retinoblastoma (Rb) family members Rb/p105 and Rb2/p130 seem to play a pivotal role in ovarian cancer. In particular, Rb/p105, when in the unphosphorylated form, acts as a growth suppressor and plays a pivotal role in the negative control of the cell cycle and in tumor progression; whereas, the phosphorylated form (p-pRB) activates genic transcription and cellular proliferation. While Rb/p105 is ubiquitously confined to the nuclei of cycling and quiescent cells, Rb2/p130 activity is also regulated by intracellular localization. According to this premise, Rb family members could represent a novel marker in diagnosis and classification risk for patients with borderline ovarian tumors (BOT).\u003c/p\u003e\u003ch2\u003eAims\u003c/h2\u003e \u003cp\u003eIn this study, we evaluated the immunohistochemical expression and subcellular localization of proteins of the retinoblastoma (Rb) gene family: Rb/p105 and Rb2/p130 in 65 ovarian borderline tumors (26 serous, 19 sero-mucinous and 20 mucinous subtypes).\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eStatistically significant differences were found in nuclear and cytoplasmic expression of Rb/p105 and Rb2/p130 according to different examined histotypes. In detail, the nuclear expression of Rb/p105 and Rb2/p130 was more frequently detected in serous (84.6%) than sero-mucinous (42.1%) and mucinous (50%) types. Conversely, the cytoplasmic expression of Rb2/p130 was not detected in serous tumors and frequently observed in mucinous subtypes (80%).\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eOur findings suggest that Rb proteins do not play a key role in the tumor progression of serous borderline tumors since they are always located in the nucleus and no cases showed a cytoplasmic localization. By contrast, the observed higher cytoplasmic expression of Rb2/p130 in mucinous BOTs (intestinal) types, is suggestive of Rb proteins involvement in the cancerogenesis pathway of mucinous ovarian tumors. Our results also suggest that mucinous BOTs of intestinal type, exhibiting low nuclear and high cytoplasmic levels of Rb2/p130 might potentially be considered a high risk category of malignant evolution. Further studies on larger series are needed in order to clarify how BOTs could be stratified in different prognostic groups according to their Rb proteins immunohistochemical profile.\u003c/p\u003e","manuscriptTitle":"Role of Retinoblastoma Protein Family (Rb/P105 And Rb2/P130) Expression in the Hystopathological Classification of Borderline Ovarian Tumors","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2020-07-28 17:34:11","doi":"10.21203/rs.3.rs-48452/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"150d9320-edd4-4f47-a8b0-addb4e20487d","owner":[],"postedDate":"July 28th, 2020","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":209150,"name":"Animal Science"}],"tags":[],"updatedAt":"2020-07-28T17:34:11+00:00","versionOfRecord":[],"versionCreatedAt":"2020-07-28 17:34:11","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-48452","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-48452","identity":"rs-48452","version":["v1"]},"buildId":"_2-kVJe1T_tPrBINL-cwx","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}