Cytoplasmic P27 Expression Research Articles

Cytoplasmic Expression of p16 Is Associated with Carcinoma Breast: It Is Not an Artifact.

p16 has different roles in the nuclear and cytoplasmic locations. The nuclear localization of the p16 protein explains its role in cell cycle regulation. Cytoplasmic expression was considered an artifact in the initial years, but there is evidence to prove that cytoplasmic localization is real and that p16 has different roles in the nuclear and cytoplasmic locations. We aimed to study the immunoexpression of p16 protein in the nuclear and cytoplasmic locations of the epithelial and stromal compartments of fibroadenoma, invasive breast carcinoma, and a select number of phyllodes tumors. The study included a total of 107 patients, comprising 51 cases of invasive breast carcinoma, 51 cases of fibroadenoma, 4 cases of benign phyllodes tumors, and 1 case of lobular carcinoma in situ (LCIS). The p16 immunohistochemistry was evaluated for nuclear and cytoplasmic localization in the epithelial and stromal compartments of the tumors. Of the 51 fibroadenoma cases, 23 showed strong nuclear p16 epithelial expression, but no case showed cytoplasmic expression. In 19/51 cases, stromal cells also showed strong p16 nuclear expression. Moderate stromal p16 expression was observed in 3 out of 4 cases of benign phyllodes. Out of the 51 cases of invasive carcinoma, 31 showed moderate to strong nuclear p16 immunopositivity, while 27 cases exhibited cytoplasmic p16 expression. We found a statistically significant correlation between moderate to strong nuclear p16 immunoexpression and the molecular subtype of breast carcinoma. The cytoplasmic localization of p16 immunohistochemistry is not seen in epithelial components of fibroadenoma, while it is seen frequently in breast carcinoma. Nuclear p16 expression has a statistically significant correlation with molecular subtypes of breast carcinoma.

Validation of a one-step genomics-based molecular classifier for endometrial carcinoma in a large Chinese population

ObjectiveThe aim of this study is to delineate the molecular classification features within Chinese endometrial cancer (EC) patients and to evaluate the concurrence between two widely employed methods for diagnosing EC molecular subtypes. MethodsThis retrospective observational cohort study encompassed 479 cases of EC for analysis. Utilizing next-generation sequencing (NGS) panels targeting POLE, TP53, and microsatellite instability (MSI) status, four subtypes [POLE ultramutated (POLE mut), MMR-deficient (MMRd), p53 abnormal (p53abn), and no specific molecular profile (NSMP)] were classified. Immunohistochemistry (IHC) was employed to ascertain the expression of p53 and MMR proteins. ResultsAmong the 479 patients, the distribution of EC subtypes was as follows: 28 (5.85%) POLE mut, 67 (13.99%) MMRd, 60 (12.53%) p53abn, and 324 (67.64%) NSMP. When compared to published findings on EC subtypes in the Caucasian population, our real-world data on Chinese ECs revealed a notably higher proportion of NSMP/CNL (copy number low). The evaluation of MSI/MMR status through NGS-based and IHC-based methods displayed substantial concordance (Kappa = 0.91). Slight discordance between the two techniques in identifying p53 abnormalities (Kappa = 0.83) might stem from TP53 truncating mutations, cytoplasmic p53 expression, null TP53 mutants, and well-documented challenges in interpreting p53 IHC. ConclusionsChinese ECs exhibit distinctive molecular attributes. For accurate molecular subtyping of Chinese ECs, additional molecular markers that align with the Chinese population's characteristics should be incorporated into existing classifiers. The study's outcomes underscore a strong agreement between NGS and IHC in TP53/p53 detection and MSI assessment.

Implication of cytoplasmic p53 expression in pulmonary neuroendocrine carcinoma using next-generation sequencinganalysis.

Cytoplasmic p53 expression indicates a high frequency of TP53 abnormalities in gynaecological carcinoma. However, the implication of this expression in pulmonary neuroendocrine carcinoma (NEC) remains unclear. Thus, our study aimed to fill this research gap. Immunohistochemistry (IHC) of p53 was performed on 146 cases of resected small-cell lung carcinoma and large-cell NEC, and next-generation sequencing was conducted on cases showing cytoplasmic and wild-type p53 expression. IHC revealed overexpression in 57% of the cases (n = 83), complete absence in 31% (n = 45), cytoplasmic expression in 8% (n = 12) and wild-type expression in 4% (n = 6) of the cases. TP53 mutations were identified in nine of the 13 cases with available genetic analysis. The TP53 mutation rates in cases with cytoplasmic and wild-type p53 expression were 88% (seven of eight) and 40% (two of five), respectively. All seven cases showing cytoplasmic expression with TP53 mutations harboured loss-of-function type mutations: four had mutations in the DNA-binding domain, two in the nuclear localisation domain and one in the tetramerisation domain. Clinically, cases with cytoplasmic p53 expression had a poor prognosis similar to that in cases with p53 overexpression or complete absence. Cytoplasmic p53 expression in patients with pulmonary NEC suggests a high TP53 mutation rate, which is associated with a poor prognosis similar to that in patients with p53 overexpression or complete absence. This cytoplasmic expression should not be misidentified as a wild-type expression. This is the first report, to our knowledge, that demonstrates the implication of cytoplasmic p53 expression in pulmonary NEC.

Expression of p16 Tumor Suppressor Protein in Malignant Ovarian Germ Cell Tumors; Immunohistochemical Study.

Malignant ovarian germ cell tumors represent small percentage of malignant ovarian neoplasms but they affect significantly young age group. To investigate the immunohistochemical expression of p16 tumor suppressor protein in malignant ovarian germ cell tumors. Twenty-two malignant ovarian germ cell tumors (five dysgerminoma, eight immature teratoma, and nine yolk sac tumors), twenty mature cystic teratoma tumors and twenty normal ovarian tissue were immunohistochemically stained with p16 monoclonal antibody. Ki67 immunohistochemical staining was done for malignant ovarian germ cell tumors to assess proliferation. We found that p16 tumor suppressor protein is overexpressed in all malignant ovarian germ cell tumors in both nuclear and cytoplasmic locations compared to control and to mature cystic teratoma (p-value <0.001). Cytoplasmic p16 expression was significantly correlated to Ki67 proliferation index in malignant ovarian germ cell tumors (p-value = 0.033, r = 0.445). Overexpression of p16 in malignant ovarian germ cell tumors denotes that dysfunction of the cyclin dependent kinase pathway is involved in tumorigenesis of malignant ovarian germ cell tumors.

Extranodal primary CD20-negative diffuse large B-cell lymphoma with cytoplasmic p16 expression

Extranodal primary CD20-negative diffuse large B-cell lymphoma (DLBCL) is a rare tumor. It often presents a diagnostic challenge for pathologists as the morphological features of neoplastic cells are similar to those of undifferentiated carcinoma or sarcoma cells. While several B-cell markers are commonly tested, expanding the panel of B-cell markers is necessary to ascertain definitive DLBCL diagnoses and would assist in decisions regarding treatment and prognoses. Several additional markers have been proposed, including several mutated genes commonly expressed in cancer cells, such as c-Myc, Bcl6/c-Myc, Bcl6, Bcl2 (double/triple expressors), as well as cytoplasmic p16.

The Zebrafish Antiapoptotic Protein BIRC2 Promotes Edwardsiella piscicida Infection by Inhibiting Caspases and Accumulating p53 in a p53 Transcription-Dependent and -Independent Manner.

IAPs (inhibitors of apoptosis) are endogenous caspase inhibitors with multiple biological activities. In the present study, we show functional characteristics of antiapoptotic protein BIRC2 (cIAP1) in response to Edwardsiella piscicida infection. Overexpression of BIRC2 in zebrafish larvae promoted the proliferation of E. piscicida, leading to a decreased larvae survival. The expression levels of caspases including casp3, casp8, and casp9 were significantly inhibited by BIRC2 overexpression in the case of E. piscicida infection. Treatment of zebrafish larvae microinjected with BIRC2 with the caspase activator PAC-1 completely blocked the negative regulation of BIRC2 on the E. piscicida infection, with the reduced inhibition on the casp3 and without inhibition on casp8 and casp9. In contrast to the regulation of BIRC2 on the caspases, BIRC2 overexpression significantly induced the expression of p53, especially at 24 hpi. In addition to the cytoplasmic p53 expression, BIRC2 overexpression also induced the expression of the nuclear p53 protein. Further analysis demonstrated that BIRC2 could interact and colocalize with p53 in the cytoplasm. The numbers of E. piscicida in larvae overexpressed with BIRC2 and treated with pifithrin-μ (an inhibitor of mitochondrial p53) or pifithrin-α (an inhibitor of p53 transactivation) were lower than those of larvae without pifithrin-μ or pifithrin-α treatment. Critically, the p53 inactivators pifithrin-μ and pifithrin-α had no significant effect on larval survival, but completely rescued larval survival for zebrafish microinjected with BIRC2 in the case of E. piscicida infection. Collectively, the present study suggest that piscine BIRC2 is a negative regulator for antibacterial immune response in response to the E. piscicida infection via inhibiting caspases, and accumulating p53 in a p53 transcription-dependent and -independent manner.

Breast Cancer and p16: Role in Proliferation, Malignant Transformation and Progression.

The definition of new molecular biomarkers could provide a more reliable approach in predicting the prognosis of invasive breast cancers (IBC). The aim of this study is to analyze the expression of p16 protein in IBC, as well as its participation in malignant transformation. The study included 147 patients diagnosed with IBC. The presence of non-invasive lesions (NIL) was noted in each IBC and surrounding tissue. p16 expression was determined by reading the percentage of nuclear and/or cytoplasmic expression in epithelial cells of IBC and NIL, but also in stromal fibroblasts. Results showed that expression of p16 increases with the progression of cytological changes in the epithelium; it is significantly higher in IBC compared to NIL (p < 0.0005). Cytoplasmic p16 expression is more prevalent in IBC (76.6%), as opposed to nuclear staining, which is characteristic of most NIL (21.1%). There is a difference in p16 expression between different molecular subtypes of IBC (p = 0.025). In the group of p16 positive tumors, pronounced mononuclear infiltrates (p = 0.047) and increased expression of p16 in stromal fibroblasts (p = 0.044) were noted. In conclusion, p16 protein plays an important role in proliferation, malignant transformation, as well as in progression from NIL to IBC.

Abstract 965: The role of LOX-mediated FAK phosphorylation in osteosarcoma metastasis

Abstract Despite the use of chemotherapy and surgery, the development of metastasis remains to be the leading cause of death in patients with osteosarcoma. LOX encodes for lysyl oxidase, which is an enigmatic molecule in cancer by acting as a tumor suppressor in certain tumors, but a metastatic promoter in other tumors. To investigate the function of LOX, we generated LOX overexpression mutants in two osteosarcoma cell lines and showed that higher LOX expression promoted tumor cell proliferation and invasiveness. Using an orthotopic xenograft mouse model, we further showed that osteosarcoma cells with higher LOX expression produced a higher number of mice with pulmonary metastases than the parental cell line (n=9). To characterize the mechanism of LOX-mediated metastasis, we examined the protein expression of a panel of potential downstream targets. The results demonstrated that phospho-FAK (focal adhesion kinase) expression was increased in the LOX-overexpression mutant relative to the parental cell line. Using a small molecule inhibitor of FAK, we showed that it significantly reduced the LOX-mediated metastasis in the orthotopic xenograft model. To demonstrate the clinical significance of our findings, we analyzed the expression profiling and RNAseq datasets recently generated from the TARGET osteosarcoma study. A Kaplan-Meier analysis showed that a higher level of LOX expression significantly correlated with a poor outcome (p &amp;lt; 0.05) and a multivariate analysis indicated LOX remained to be significant after controlling for initial metastatic status. In addition, we have previously shown that p27 is frequently mislocalized in the cytoplasm of osteosarcoma cells and drives the development of metastasis through interacting with the PAK1-mediated actin cytoskeleton network. Our results showed that the expression of LOX was upregulated in p27-mislocalized osteosarcoma cell lines with high metastatic potential. A tissue microarray analysis showed that cytoplasmic p27 expression significantly correlated with LOX expression (p = 0.003). Together, our findings suggest that a novel link between p27 mislocalization and LOX expression. LOX plays a pivotal role in osteosarcoma metastasis by upregulating FAK phosphorylation. FAK inhibition is a potential therapeutic strategy to reduce the development of metastasis in osteosarcoma with LOX overexpression. Citation Format: Xiang Chen, John M. Hicks, Poonam Sarkar, Waleed M. Gaber, Tsz-Kwong MAN. The role of LOX-mediated FAK phosphorylation in osteosarcoma metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 965.

Effects of Cisplatin on Superoxide Anion, Sod, Apoptosis and Cytoplasmic p21Expression in C666-1 Cell Lines Undifferentiated Nasopharyngeal Cancer

Effects of Cisplatin on Superoxide Anion, Sod, Apoptosis and Cytoplasmic p21Expression in C666-1 Cell Lines Undifferentiated Nasopharyngeal Cancer

Increased Cytoplasmic Yes-associated Protein (YAP) Expression in Mismatch Repair Protein-Proficient Colorectal Cancer With High-grade Tumor Budding and Reduced Autophagy Activity.

Yes-associated protein (YAP) is a transcriptional coactivator regulated by autophagy that stimulates colorectal cancer (CRC) progression through activation of epithelial-mesenchymal transition (EMT), represented by tumor budding. The associations between these components in CRC are unknown. Archived surgically resected CRCs with known mismatch repair protein (MMR) status were retrieved (n=81; 2010 to 2016). Electronic medical records were reviewed for clinicopathologic variables including pathologic TNM stage and clinical stage. Tumor budding was graded according to consensus guidelines. Cytoplasmic and nuclear YAP and p62 (autophagy substrate) immunoreactivity were semiquantitatively scored within tumor samples. The Student t test, Fisher exact test, χ2 test, and Spearman correlation coefficient were performed with P<0.05 as a significance level. MMR proficiency (MMR-P) status correlated with high-grade tumor budding. The extent of cytoplasmic YAP staining and pathologic N stage was associated with tumor budding in multivariate analysis. Cytoplasmic YAP expression correlated with higher cytoplasmic p62 expression, suggesting an inverse correlation between autophagy activation and cytoplasmic YAP expression. Nuclear YAP expression correlated with pathologic N stage and clinical stage. A correlation between MMR-P status and tumor budding, combined with correlations between cytoplasmic YAP, tumor budding and p62 raise the possibility of 2 distinct neoplastic pathways concerning autophagy and YAP; one displaying relative activation of YAP and EMT, being commonly observed in MMR-P, and another with less active YAP and EMT, but active autophagy, being commonly seen in MMR-deficient CRC. Nuclear YAP staining could be useful in prognostication.

Autophagy modulates mesenchymal-to-endothelial transition via p53.

Mesenchymal-to-endothelial transition (MEndT) is one of the mechanisms that influences cardiac fibrosis, which is a key process in cardiac remodeling. It has been reported that autophagy inhibits endothelial cell transition. However, whether autophagy could modulate MEndT in cardiac fibrosis has not yet been investigated. Here, we discussed the association between autophagy and MEndT and its possible mechanism. In this study, we induced endothelial-to-mesenchymal transition using transforming growth factor-β to generate mesenchymal cells and fibroblasts in wild-type human umbilical vein endothelial cells and cells with p53 knockout or overexpression. Then, autophagy was induced by Earle's balanced salt solution (EBSS) and was inhibited by bafilomycin A1 or lentivirus-ATG5-shRNA. The expression levels of MEndT and the autophagy markers CD31, VE-Cadherin, Vimentin, α-SMA, LC3, p62 and p53 were examined. We found that activation of autophagy could promote MEndT and increase cytoplasmic and total expression of p53, that but nuclear p53 expression was decreased, and that inhibition of autophagy activation could reverse the effect of EBSS. Moreover, after knockout of nuclear p53, autophagy promoted MEndT, while autophagy inhibited MEndT in p53 overexpressing cells. Our results demonstrate that autophagy modulate MEndT by nuclear p53 provide a new strategy for the treatment of fibrosis diseases.

Cytological interpretation of p16 immunohistochemistry in head and neck carcinomas: does the choice of fixative matter?

Fine-needle aspiration (FNA) of nodal metastases plays a key role in the diagnosis of oropharyngeal squamous cell carcinoma (OPSCC). Because of significant clinical implications of human papillomavirus (HPV)-related OPSCC, immunohistochemistry for p16 as a surrogate marker for high-risk HPV is an important ancillary test. After our laboratory switched from CytoLyt to formalin fixative for FNA needle rinses generating cell block (CB) material, we investigated the impact of this protocol change on the accuracy of p16 results. FNA specimens of head and neck lesions with p16 staining performed on CB, from 1 year before and after the implementation of formalin-fixed CB (FCB) were identified. Nuclear and cytoplasmic p16 expression was scored and compared to p16 status on corresponding surgical specimens. There were no false-positive results with either fixative. CytoLyt-fixed CB (CCB) had 47% (7 of 15) false-negative cases, whereas FCB had none, with 100% diagnostic accuracy for p16-negative (n = 6) and p16-positive (n = 15) results. False-negative CCB showed 0% to 10% nuclear and 0% to 65% weak cytoplasmic staining, whereas true-positive CCB showed 10% to 85% nuclear and 35% to 90% cytoplasmic staining. p16-negative FCB showed 0% nuclear and cytoplasmic staining, and p16-positive FCB showed 30% to 100% moderate-strong nuclear and cytoplasmic staining. Interobserver variability was greater with CCB. In our laboratory, formalin fixation of CB material improved the accuracy of p16 interpretation. Staining in FCB was also more robust than CCB, which showed weaker cytoplasmic and more focal nuclear staining. Therefore, we advocate formalin fixation for head and neck cytology specimens that may require p16 testing.

Abstract 747: Pak1, TGFα, Ki 67 and survival in human gastric and gastroesophageal junction adenocarcinomas

Abstract Stomach cancer is the third most common cause of cancer mortality worldwide. Most are adenocarcinomas. Adenocarcinomas of the stomach fall into two obvious groups based on site of origin; those arising in the area of the gastroesophageal junction (GEJ) and those arising from a more distal location, which we term gastric in this study. Adenocarcinomas of the stomach fall into four obvious groups based on a combination of genomic, molecular biologic, clinical and histopathologic characteristics (Nagara A, Kikuchi O, Bass AJ, Cancer Discovery 2019; DOI: 10.1158/2159-8290.CD-19-0487). We are reporting herein a study of 99 patients with adenocarcinoma of the stomach. This study evaluates the location of the tumor, the histopathologic features of the tumor, the immunohistochemically determined nuclear and cytoplasmic expression of P21 activated kinase (Pak 1), Transforming Growth Factor α (TGFα), and Ki67, a marker of cell proliferation. This study correlates these findings with patient survival. The study reveals: 1. significantly lower survival in patients with diffuse type adenocarcinoma compared to intestinal type, in both GEJ and gastric locations. 2. both GEJ and gastric adenocarcinomas had a poor prognosis. 3. increased expression of both TGFα and Pak1 in GEJ adenocarcinomas. 4. Ki67 expression is significantly less in diffuse type than the intestinal type (p=0.01 for GEJ and 0.03 for gastric). 5. cytoplasmic Pak1 and cytoplasmic TGFα were highly positively correlated (p=0.0001). 6. Ki67 expression was significantly inversely correlated with both nuclear Pak1 (p=0.04) and nuclear TGFα (p=0.02). 7. significantly increased expression of nuclear Pak1 in gastric diffuse type compared with gastric intestinal (p=0.0002) and GEJ intestinal (p=0.0001). These data suggest that Pak1 and TGFα might serve as diagnostic biomarkers of subtypes of adenocarcinomas of the stomach and gastroesophogeal junction. Citation Format: Larry E. Douglass, Michael Parritt, Adam Neltner, Mariah Dooley, Michelle Robillard, Molly Frydl, Leah Focke, Kyle Damen, James A. Deddens, Julia H. Carter. Pak1, TGFα, Ki 67 and survival in human gastric and gastroesophageal junction adenocarcinomas [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 747.

A subset of sinonasal undifferentiated carcinoma is associated with transcriptionally active high-risk human papillomavirus by in situ hybridization: a clinical and pathologic analysis

Sinonasal undifferentiated carcinoma (SNUC) is an aggressive malignancy with a poor prognosis, and pathologically, it is a diagnosis of exclusion. Rendering this diagnosis can be challenging in practice because of the large number of diverse entities in the differential diagnosis. We encountered an index case of a sinonasal carcinoma otherwise diagnosable as SNUC which, on further investigation, demonstrated strong and diffuse P16 expression, as well as diffuse expression of high-risk human papillomavirus (hrHPV) RNA by in situ hybridization (ISH). We therefore hypothesized that a subset of cases previously diagnosed as SNUC may in fact harbor transcriptionally active hrHPV. We further investigated a cohort of 25 SNUC cases in our pathology archivesand performed ISH for hrHPV RNA on cases that demonstrated >70% nuclear and cytoplasmic P16 expression, criteria which, in other anatomic sites, correlates strongly with the presence of hrHPV. Twelve of 25 SNUC cases were P16 positive, and of these, 5 were positive for hrHPV by ISH. Thus, 20% of all SNUC cases in this cohort harbored transcriptionally active hrHPV. Herein, we report a clinical and pathologic analysis of these cases, including differential diagnostic considerations and comparison of their clinical behavior with SNUC cases that are negative for hrHPV by ISH.

Cytoplasmic p27 is a novel prognostic biomarker and oncogenic protein for nasopharyngeal carcinoma

Cytoplasmic p27 plays an important role in regulating the cell cycle. Recent studies have revealed p27 protein translocation from the nucleus to the cytoplasm in many tumour cells. The aim of this study was to investigate the role and molecular mechanisms of cytoplasmic p27 in the progression of nasopharyngeal carcinoma (NPC) and to explore its prognostic value. We found increased cytoplasmic p27 expression by immunohistochemistry in NPC tissues, and its expression level was significantly correlated with the T classification and TNM clinical stage of NPC. The survival rate was significantly lower for NPC patients with cytoplasmic p27 immunopositivity than for NPC patients with cytoplasmic p27 immunonegativity, and cytoplasmic p27 was an independent risk factor that affected the prognosis of patients with NPC. Cytoplasmic p27 promoted the proliferation, cell cycle progression, migration, and invasion of NPC cells, increased Bim-1 and Twist1 protein levels, and decreased RhoA-GTP level. Collectively, these findings suggest that cytoplasmic relocalization of p27 is involved in the pathogenesis of NPC and is closely related to the unfavourable prognosis of patients with NPC. Therefore, cytoplasmic p27 might be a useful prognostic factor and potential therapeutic target for patients with NPC.

Prognostic Significance of LC3B and p62/SQSTM1 Expression in Gastric Adenocarcinoma.

Autophagy is a cellular mechanism that recycles cellular components to maintain homeostasis. To investigate the clinical implication of autophagy in gastric cancer, the autophagy markers with autophagosome formation, LC3B and selective autophagy substrate p62/SQSTM1 (P62) were validated. LC3B and p62 expression was examined using immunohistochemistry, western blot assays, and reverse-transcription polymerase chain reaction (RT-PCR). The relationship of LC3B and p62 expression in gastric adenocarcinomas with clinicopathological parameters, including patient survival, were analyzed. Normal gastric mucosae exhibit no LC3B and p62 expression, while tubular adenoma and gastric adenocarcinomas exhibit variable nuclear or cytoplasmic p62 expression. High LC3B, high cytoplasmic p62, and low nuclear p62 protein expression in gastric adenocarcinomas is positively correlated with poor prognostic factors including survival. Dynamic LC3B and p62 changes are suggested to be involved in gastric tumorigenesis and cancer progression. LC3B and p62 could be used as prognostic biomarkers and potential therapeutic targets for gastric adenocarcinomas.

Abstract GMM-045: PAX8 PLAYS AN ESSENTIAL ROLE IN HIGH GRADE SEROUS OVARIAN CANCER VIA ACTIVATION OF MUTANT P53 AND MISLOCALIZED P21

Abstract High grade serous carcinoma (HGSC) is the most common subtype of ovarian cancer, and a very deadly disease, mostly due to lack of early detection methods. Wide spread disease at diagnosis has delayed the understanding that it often originates from the fallopian tube. This novel understanding has led to increased interest in fallopian tube developmental lineage markers, such as PAX8. In the adult fallopian tube, PAX8 is expressed in the fallopian tube secretory epithelial cell (FTSEC) and its expression is maintained through the process of FTSEC transformation Serous Tubal Intraepithelial Carcinoma (STIC) and to HGSC. We show that PAX8 has an essential pro-proliferative and anti-apoptotic role in HGSC. This role is mediated through direct transcriptional activation of mutated TP53, which often carries missense mutations that potentially lead to gain of function (GOF) oncogenic activities. Surprisingly, mutant p53 binds the p21 promoter and transcriptionally activates the expression of p21, which localizes to the cytoplasm of cells where it plays a non-canonical, pro-proliferative role. While in normal fallopian tube p21 is nuclear as expected, and STIC shows a mix of nuclear and cytoplasmic staining, an analysis of p21 staining in EOC shows that cytoplasmic p21 expression correlates with bad prognostic factors. Together, our findings illustrate that in HGSC TP53 mutations and abnormal p21 localization subvert a normal developmental pathway into a driver of tumor progression. Citation Format: Dima Ghannam, Eyal Jacob, Reli Kakun, Tanya Wasserman, Lina Korsensky, Ofir Sternfeld, Julia Kagan, Debora Rosa Bublik, Sarit Aviel-Ronen, Keren Levanon, Edmond Sabo, Sarit Larisch, Moshe Oren, Dov Hershkovitz, Ruth Perets. PAX8 PLAYS AN ESSENTIAL ROLE IN HIGH GRADE SEROUS OVARIAN CANCER VIA ACTIVATION OF MUTANT P53 AND MISLOCALIZED P21 [abstract]. In: Proceedings of the 12th Biennial Ovarian Cancer Research Symposium; Sep 13-15, 2018; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2019;25(22 Suppl):Abstract nr GMM-045.

Mutational and Immunophenotypic Profiling of a Series of 8 Tubo-ovarian Carcinosarcomas Revealed a Monoclonal Origin of the Disease.

Carcinosarcomas are rare, highly aggressive neoplasms composed of a combination of carcinomatous and sarcomatous elements. These tumors represent a paradigmatic field for the study of intratumor heterogeneity. A series of 8 tubo-ovarian carcinosarcomas was characterized for the following: (i) immunohistochemical expression of MNF116, epithelial membrane antigen, vimentin, S100, chromogranin, synaptophysin, desmin, myogenin (MYF4), and p53; (ii) mutational profiling of KRAS, BRAF, PIK3CA, NRAS, TP53, and DICER1 genes. Heterologous differentiation was present in 6 of 8 tumors. Cytokeratin MNF116 and epithelial membrane antigen were positive in all the carcinomatous components and in 87.5% and 50.0% of the sarcomatous components, respectively. The sarcomatous components showed positive staining for vimentin in all cases. Two cases demonstrated positivity for neuroendocrine markers in their carcinomatous components. All rhabdomyosarcomas were positive for desmin and MYF-4. Chondrosarcomas were positive for S100. All but one tumor showed similar p53 immunoreactivity in both the carcinomatous and sarcomatous components, and one case showed cytoplasmic p53 expression. Three of 8 cases (37.5%) showed TP53 mutations, and, in 2 cases, the TP53 mutation was shared by both epithelial and mesenchymal components. DICER1 mutation was found in all components of one case. Mutations in KRAS, NRAS, BRAF, and PIK3CA genes were not found in the study cohort. Our results highlight the heterogeneity of ovarian carcinosarcomas at the phenotypic level. A common mutational signature was observed in both components in 3 of 4 informative tumors. More studies are required to dissect different levels of ovarian carcinosarcomas' heterogeneity in order to define the best therapeutic approaches to these aggressive neoplasms.

P16 Expression Correlates with Invasive Ocular Surface Squamous Neoplasms in HIV-Infected Mozambicans

Background: p16 immunohistochemistry is widely used to diagnose human papillomavirus (HPV)-related squamous neoplasms of cervix, anogenital, head, and neck tissues. The incidence of these HPV-related squamous neoplasms is markedly increased in the HIV-infected population. Ocular surface squamous neoplasia (OSSN) is also more common in HIV-infected patients. However, the expression pattern of p16 in OSSN among HIV-infected patients is unclear. Here, we examined the expression of p16 in OSSN surgical excisions collected from a large HIV-infected cohort from ­Mozambique. Methods: OSSN surgical tissue specimens were collected from 75 Mozambican patients. Formalin-fixed, paraffin-embedded tissue blocks from these OSSNs were sectioned, stained with hematoxylin and eosin (H&E), and p16 expression by immunohistochemistry. H&E slides were reviewed to determine if OSSNs were noninvasive conjunctival intraepithelial neoplasms or invasive squamous cell carcinomas (SCC). Cases were classified as p16 positive or negative based on diffuse nuclear and cytoplasmic expression of p16 in neoplastic cells. Results: p16 positivity was found in a minority of OSSN cases (14/75). p16 positivity was significantly associated with the invasive SCC type of OSSN in HIV-infected patients (p value of 0.026). Conclusions: The majority of OSSNs in our HIV-infected cohort do not express p16. However, those cases that are p16-positive are significantly more likely to be the invasive SCC form of OSSN. We propose that p16 expression may identify more aggressive OSSNs in HIV-infected populations.

Abstract LB-230: Refining treatment recommendations for lymph node-negative breast cancer patients using a novel protein-based prognostic signature: The OncoMasTR assay

Abstract Introduction Traditionally, many women with lymph node-negative breast cancer (LN-BC) are unnecessarily treated with chemotherapy. This highlights the importance of accurately predicting patient prognosis. We previously identified a novel prognostic signature encompassing a set of master transcriptional regulators (MTRs), termed OncoMasTR. At the mRNA level, OncoMasTR predicted recurrence risk for LN-BC better than current prognostic signatures and accurately classifies &amp;gt;50% of patients as ‘low-risk', thus potentially reducing overtreatment. Here, we describe ongoing validation of the OncoMasTR biomarker signature at the protein level. Methods Commercially available antibodies targeted against markers were validated by Western blotting and immunohistochemistry (IHC) using CRISPR/Cas9 knockout cell-lines and full-face BC sections. IHC was performed on tissue microarrays (TMAs) containing multiple tumour cores from BC patients. Visiopharm's Oncotopix algorithm was used for image analysis, with stained sections verified manually by a pathologist. Image analysis data was obtained, and cut-off points optimised by maximum Chi-square values were used to differentiate low and high expression of individual markers. This was combined with clinical data to generate Kaplan-Meier survival curves. Multivariate analysis was used to determine the optimal combination of biomarkers. Results Antibodies against eight components of the OncoMasTR signature have been validated. IHC analysis demonstrated highly-specific nuclear staining for seven of the markers (UHRF1, ATAD2, HMGB2, E2F1, TCF19, MYBL2, PTTG1), whilst one showed both nuclear and cytoplasmic staining (p16). Image analysis for UHRF1, ATAD2 and HMGB2, using both manual and automated histological scores, showed that high expression of these biomarkers is linked with reduced survival. Additionally, UHRF1 was confirmed to have the same survival correlation in another independent cohort. Interestingly, high nuclear expression of p16 is linked with prolonged survival whilst high cytoplasmic expression of p16 is associated with poorer survival, aligning with previous reports. Conclusions The OncoMasTR signature offers a more accurate stratification of low-vs-high risk, thus showing promise in improving prognosis prediction for early-stage BC. This will translate to reduced overtreatment, decreased costs, improved survival rates and enhanced quality of life for patients. Citation Format: Arman Rahman, Seodhna Lynch, Nebras Alattar, Niamh Niamh, Charles Weige,, Romina Silva, Claudia Aura, Fiona Lanigan, Adrian Bracken, Björn Nodin, Karin Jirström, William Gallagher. Refining treatment recommendations for lymph node-negative breast cancer patients using a novel protein-based prognostic signature: The OncoMasTR assay [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-230.