Endometrial Clear Cell Carcinoma Research Articles

EP-1321: Postoperative treatment results of clear-cell endometrial carcinoma: 20 cases from 2005 to 2014

EP-1321: Postoperative treatment results of clear-cell endometrial carcinoma: 20 cases from 2005 to 2014

Abstract B12: Targeted sequencing of ovarian clear cell carcinomas reveals intertumor heterogeneity and distinct clustering related to mutational load.

Abstract Objective: Ovarian clear cell carcinomas (OCCCs) constitute a rare ovarian cancer subtype. Despite relatively clear clinical features, OCCCs can be difficult to distinguish morphologically from other epithelial ovarian cancers. OCCC gene expression signatures capturing not only the morphology but also the biology of this tumor subtype have been described, and a previous study using comparative genomic hybridization showed that OCCCs are indeed heterogeneous. Still, only a few genes have been analyzed using gene sequencing techniques. In the present study we aimed to further characterize OCCCs and their histotype specific genetic changes using targeted sequencing of 60 well-established cancer genes. Increased knowledge about genetic aberrations in OCCCs may help to guide future development of targeted treatments and improve the outcome for these patients. Methods: Formalin fixed, paraffin-embedded (FFPE) tumor tissue was collected from 11 primary OCCCs, diagnosed at the Skane University Hospital, Lund, Sweden (1998-2013). One sample was excluded due to poor DNA quality. For the remaining 10 samples, 600 ng genomic DNA/sample was used for single strand targeted DNA sequencing using the SureSeq™ solid tumor panel consisting of 60 key genes involved in cancer evolution, including ovarian cancer. The panel covers all coding exons in the included genes, and is validated for research use on FFPE samples. The publicly available ENSEMBL database was used to classify the mutations according to severity across the genes. Serious mutations, defined as major non-conservative amino acid changes, splice or frameshift variants or changes in stop codons, affecting single nucleotide variations (SNVs) or insertions or deletions of DNA bases (indels) were used for further analyses. The mutation call cut-off was set to 20% and the reading depth ≥300x, and unsupervised hierarchical clustering of the tumor samples was performed. Results: >99% of the target bases had a 30x coverage across all samples, and the mean target coverage was 534x. Using the 20% cut-off, approximately 300 mutations were detected in the 60 genes across the 10 tumor samples. Unsupervised hierarchical clustering revealed two distinct clusters related to mutational load. Mutations in genes previously reported to be affected in OCCCs, such as ARID1A and ERBB2, were identified. Whereas ARID1A was mainly affected in one cluster, mutations in genes signifying a highly malignant phenotype, such as BRCA1 and BRCA2 dominated in the other. Genes in signaling pathways often affected in malignant tumors, as e.g. the RAS pathway, however, were unaffected. Other mutations detected in this dataset were ZFHX3 and KMT2C. KMT2C was the only highly mutated gene across both clusters. The clustering pattern was not related to age of FFPE blocks or tumor cell content. Conclusions: In the present study we show that OCCCs are genetically heterogeneous. The variation in mutation rate is in line with a previous publication, and points to differences in tumor biology that may be important for tumor behavior and thus patient outcome. The highly mutated genes may help in guiding trials of targeted treatments; the methyl transferase gene KMT2C, also known as MLL3, may potentially be one such target. Rearrangements of this gene are well-described in acute leukemias. Interestingly, KMT2C/MLL3 is also reported to be a mutational cancer driver gene in renal clear cell carcinomas according to the TCGA. Likewise, ZFHX3 has recently been reported in endometrial clear cell carcinomas. Despite the small size of this study it points to the well-established fact that ovarian cancer is not one single disease, and also confirms that even the rare OCCCs seem to harbor different biological features which may be of potential clinical importance. Citation Format: Jenny-Maria Jönsson, Nicolai Skovbjerg Arildsen, Anna Ebbesson, Sofia Westbom-Fremer, Anna Måsbäck, Susanne Malander, Mef Nilbert, Ingrid Hedenfalk. Targeted sequencing of ovarian clear cell carcinomas reveals intertumor heterogeneity and distinct clustering related to mutational load. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr B12.

Hypercalcemia Due to Parathyroid Hormone-Related Protein Induced by Primary Endometrial Clear Cell Adenocarcinoma: Case Report

Humoral Hypercalcemia of Malignancy (HHM) has been reported in association with a number of malignancies. In gynecologic malignancies, ovarian Clear Cell Carcinoma (CCC) is one of the most commonhistologic subtypes, whereas HHM caused by endometrial CCC is very rare. We report a case of endometrial CCC with HHM, with a low serum intact PTH level, elevated serum PTH-related Peptide (PTH-rP), and immunohistochemically demonstrated PTH-rP in the neoplasm.

Expression, Epigenetic and Genetic Changes of HNF1B in Endometrial Lesions.

Hepatocyte nuclear factor 1-beta (HNF-1-beta) is a transcription factor involved in cancerogenesis of various tumors, including endometrioid carcinoma. We performed comprehensive analysis of HNF-1-beta in lesions of the endometrium, including protein expression and genetic and epigenetic changes. Expression of HNF-1-beta was analyzed immunohistochemically in 320 cases including both tumor and non-tumor endometrial lesions. Promoter methylation and genetic variants were evaluated, using bisulphite and direct sequencing, in 30 (18 fresh frozen, 12 FFPE tumors) endometrioid carcinomas (ECs) and 15 ovarian clear cell carcinomas (OCCCs) as a control group. We detected expression of HNF-1-beta in 28% of ECs (51/180 cases), 26% of serous carcinoma (7/27 cases), 83% of endometrial clear cell carcinoma (15/18 cases), 93% of hyperplastic polyps with atypias (13/14 cases), 100% of hyperplastic polyps without atypias (16/16 cases), 88% of hyperplasias with atypias (14/16 cases), 91% of hyperplasias without atypias (10/11 cases), and in ≥80% of different normal endometrium samples. The control group of OCCCs showed HNF-1-beta expression in 95% (18/19 cases). Methylation in promoter region was detected in 13.3% (4/30) of ECs, but not in corresponding normal tissue where available, nor in OCCCs (0/15 cases). Mutation analysis revealed truncating variant c.454C>T (p.Gln152X) in one EC and missense variant c.848C>T (p.Ala283Val) was detected in one OCCC. In conclusion, expression of HNF-1-beta was detected in various extents in all types of lesions analyzed, nevertheless its strong expression was mostly limited to clear cell carcinomas. Biological significance of genetic and epigenetic changes needs further investigation.

Survival Outcomes in Patients With Clear Cell Endometrial Carcinoma Are Improved With Surgery Plus Adjuvant Radiation Therapy: A Population-Based Study

Survival Outcomes in Patients With Clear Cell Endometrial Carcinoma Are Improved With Surgery Plus Adjuvant Radiation Therapy: A Population-Based Study

Impact of Adjuvant Radiation Therapy and Chemotherapy on Stage I-II Clear Cell Endometrial Carcinoma: A National Cancer Data Base (NCDB) Analysis

Impact of Adjuvant Radiation Therapy and Chemotherapy on Stage I-II Clear Cell Endometrial Carcinoma: A National Cancer Data Base (NCDB) Analysis

Endometrial Carcinomas With Clear Cells: A Study of a Heterogeneous Group of Tumors Including Interobserver Variability, Mutation Analysis, and Immunohistochemistry With HNF-1β.

Endometrial clear cell carcinoma (CC) is an uncommon tumor and often carries a poor prognosis. It has histologic features that overlap with other endometrial carcinomas and is frequently misclassified. Accurate classification is crucial, however, to improve treatment options. The objectives of this study were (1) to assess diagnostic interobserver variability among 5 gynecologic pathologists for tumors originally diagnosed as CC or with a component of CC (n=44); (2) to determine the utility of immunohistochemical markers estrogen receptor and HNF-1β; and (3) to detect mutations in select genes. Clinical data and morphologic features were also recorded. Agreement among reviewers was only moderate: only 46% of the original CC remained classified as such. After reclassification, estrogen receptor was positive in 8% of CC, 67% of endometrioid carcinomas (EC), and 47% of serous carcinomas (SC). Sensitivities of HNF-1β in CC, SC, and EC were 62%, 27%, and 17%, respectively, whereas specificity for CC versus EC or SC was 78%. Mutations in PIK3CA, PIK3R1, PTEN, KRAS, and NRAS were detected in 41% of 37 cases that had adequate material for study. At least 1 mutation was identified in 33% of CC, 67% of EC, and 33% of SC. This group of patients had poor outcomes: 72% of the patients with follow-up information had died of disease. In summary, this study suggests that the current pool of CC is a heterogeneous group of tumors from the morphologic, immunophenotypic, and molecular point of views and that only a percentage of them represent true CC.

Are clear cell carcinomas of the ovary and endometrium phenotypically identical? A proteomic analysis.

Phenotypic differences between otherwise similar tumors arising from different gynecologic locations may be highly significant in understanding the underlying driver molecular events at each site and may potentially offer insights into differential responses to treatment. In this study, the authors sought to identify and quantify phenotypic differences between ovarian clear cell carcinoma (OCCC) and endometrial clear cell carcinoma (ECCC) using a proteomic approach. Tissue microarrays were constructed from tumor samples of 108 patients (54 ECCCs and 54 OCCCs). Formalin-fixed samples on microarray slides were analyzed by matrix-assisted laser desorption/ionization mass spectrometry, and 730 spectral peaks were generated from the combined data set. A linear mixed-effect model with random intercept was used to generate 93 (12.7%) peaks that were significantly different between OCCCs and ECCCs at the fold cutoffs of 1.5 and 0.667 and an adjusted P value cutoff of 1.0 × 10(-10). Liquid chromatography-tandem mass spectrometry was performed on selected cores from each group, and peptides identified therefrom were compared with lists of statistically significant peaks from the aforementioned linear mixed-effects model to find matches within 0.2 Da. A total of 53 candidate proteins were thus identified as being differentially expressed in OCCCs and ECCCs, 45 (85%) of which were expressed at higher levels in ECCCs than OCCCs. These proteins were functionally diverse and did not highlight a clearly dominant cellular theme or molecular pathway. Although ECCCs and OCCCs are very similar, some phenotypic differences are demonstrable. Additional studies of these differentially expressed proteins may ultimately clarify the significance of these differences.

Molecular profile comparison of endometrial, renal and ovarian clear cell carcinoma: Is it the same disease at different sites?

5595 Background: Clear cell carcinomas (CCC) are histologically similar, however their clinical course varies widely based on the organ of origin. Clear cell uterine carcinoma (CCUC) accounts for a...

An international comparison of surgically staged patients with serous and clear cell endometrial carcinoma

An international comparison of surgically staged patients with serous and clear cell endometrial carcinoma

Molecular alterations in endometrial and ovarian clear cell carcinomas: clinical impacts of telomerase reverse transcriptase promoter mutation

Recently, mutations of telomerase reverse transcriptase (TERT) promoter were found in several types of cancer. A few reports demonstrate TERT promoter mutations in ovarian clear cell carcinomas but endometrial clear cell carcinoma has not been studied. The aims of this study were to compare differences of molecular alterations and clinical factors, and identify their prognostic impact in endometrial and ovarian clear cell carcinomas. We evaluated mutations of the TERT promoter and PIK3CA, expression of ARID1A, and other clinicopathological factors in 56 ovarian and 14 endometrial clear cell carcinomas. We found that TERT promoter mutations were present in 21% (3/14) of endometrial clear cell carcinomas and 16% (9/56) of ovarian clear cell carcinomas. Compared with ovarian clear cell carcinomas, endometrial clear cell carcinomas showed older mean patient age (P<0.001), preserved ARID1A immunoreactivity (P=0.017) and infrequent PIK3CA mutation (P=0.025). In ovarian clear cell carcinomas, TERT promoter mutations were correlated with patient age >45 (P=0.045) and preserved ARID1A expression (P=0.003). In cases of endometrial clear cell carcinoma, TERT promoter mutations were not statistically associated with any other clinicopathological factors. In ovarian clear cell carcinoma patients with early FIGO stage (stages I and II), TERT promoter mutation was an independent prognostic factor and correlated with a shorter disease-free survival and overall survival (P=0.015 and 0.009, respectively). In recurrent ovarian clear cell carcinoma patients with early FIGO stage, TERT promoter mutations were associated with early relapse within 6 months (P=0.018). We concluded that TERT promoter mutations were present in endometrial and ovarian clear cell carcinomas. Distinct molecular alteration patterns in endometrial and ovarian clear cell carcinomas implied different processes of tumorigenesis in these morphologically similar tumors. In ovarian clear cell carcinoma of early FIGO stage, patients with TERT promoter mutation require close follow-up during the initial 6 months following chemotherapy.

Targeted mutation analysis of endometrial clear cell carcinoma.

Endometrial clear cell carcinomas (CCC) constitute fewer than 5% of all carcinomas of the endometrium. Currently, little is known regarding the genetic basis of endometrial CCC. We performed genomic and immunohistochemical analyses on 14 rigorously reviewed pure endometrial CCC. The genomic analysis consisted of sequencing the coding regions of 26 genes implicated previously in endometrial carcinoma. Twelve of 14 tumours displayed a prototypical CCC immunophenotype [napsin A+, hepatocyte nuclear factor-1β (HNF1β(+) ) and oestrogen receptor(-) ] and all showed intact mismatch repair protein expression. We detected mutations in 11 of 14 tumours, and there was a predominance of mutations involving genes that are mutated more frequently in endometrial serous carcinomas than in endometrioid carcinomas. Two tumours displayed a prototypical serous carcinoma mutation profile (concurrent TP53 and PPP2R1A mutations, without PTEN, CTNNB1 or ARID1A mutation). No mutations in PTEN, CTNNB1 or POLE were identified. The overall mutation profile of this cohort of endometrial CCC appears to be more serous-like than endometrioid-like, with a minor subset in the TP53-mutated CCC showing serous carcinoma profile. These findings provide new insights into the molecular features of morphologically prototypical endometrial CCC, and underscore the need for further investigations into the oncogenesis of endometrial CCC.

Early-Stage Pure and Mixed Endometrial Clear Cell Carcinoma: Outcomes and Patterns of Recurrence

Clear cell carcinoma (CCC) is a rare form of uterine cancer with propensity for extrauterine spread and aggressive behavior. Standard of treatment for early stage disease is unclear. Our study focused on investigating outcomes and patterns of failure in patients with stage I-II endometrial CCC.

Abstract number 1: The futility of transvaginal ultrasound in type II endometrial cancer

number 1: The futility of transvaginal ultrasound in type II endometrial cancer C. Billingsley, K. Kenne, C. Cansino, D. O'Malley, D. Cohn, J. Fowler, E. Eisenhauer, L. Copeland, F. Backes, R. Salani, The Ohio State University, Columbus, OH, USA, University of California Davis, Sacramento, CA, USA, University of Cincinnati, UC Health Medical Arts Building, Cincinnati, OH, USA Objectives: To determine the utility of transvaginal ultrasound (TVUS) in women with type II endometrial cancer, including serous, clear cell, and high-grade carcinomas. Methods: A retrospective review was conducted in women with pathology proven type II endometrial cancer that underwent preoperative TVUS. The following data were obtained: endometrial stripe (EMS) measurement, presence of intracavitary fluid or lesion, myometrial and/or adnexal masses, uterine size and volume. Pathology reports were reviewed and clinicopathologic factors were abstracted. Descriptive analyses were performed to assess demographic and comparative data. Results: Sixty-three women comprised the study cohort and the median age was 65 years. The most commonly reported symptom was postmenopausal bleeding in 51 patients (80.9%). The EMS was reported as thin (b5 mm) or indistinct in 18 patients (28.6%). Approximately 60% of patients were noted to have one or more ultrasound abnormalities: intracavitary lesion (18, 28.6%), intracavitary fluid (8, 12.7%), myometrial lesion (19, 30.2%), and adnexal mass (8, 12.7%). Poorly differentiated endometrioid cancer (33; 52.4%) represented the predominant histology. Of the 28% of women with a thin/indistinct EMS, seven women did not have an ultrasound abnormality, representing 11% of women with type II endometrial cancer who had no abnormal ultrasound findings whatsoever. Conclusions: Women with type II endometrial cancer were found to have a thin/indistinct EMS on TVUS in approximately 25% of cases. Furthermore, lack of any ultrasound abnormality, including a thickened EMS, were noted in 11% of patients. The use of TVUS, which has been of diagnostic and triaging value for women with type I cancer, is of limited utility in type II endometrial cancer. Therefore, endometrial sampling should be included in the evaluation of all women with postmenopausal bleeding, regardless of EMS thickness. doi:10.1016/j.ygyno.2014.04.019 Abstract number 2: Durationof intra-chemotherapy anemia is associatedwith prognosis and survival in epithelial ovarian cancer R. Brightwell, K. Eng, S. Lele, K. Odunsi, Roswell Park Cancer Institute,number 2: Durationof intra-chemotherapy anemia is associatedwith prognosis and survival in epithelial ovarian cancer R. Brightwell, K. Eng, S. Lele, K. Odunsi, Roswell Park Cancer Institute,

Survival of women with clear cell and papillary serous endometrial cancer after adjuvant radiotherapy.

BackgroundType II (papillary serous and clear cell) endometrial carcinoma (EC) is a rare subgroup and is considered to have an unfavorable prognosis. The purpose of this retrospective analysis was to elucidate the meaning of adjuvant radiotherapy (RT) for clinical outcome and to define prognostic factors in these patients (pts).MethodsFrom 2004-2012 forty-two pts with type II EC underwent surgery followed by adjuvant RT at our department. Median age was 72 years. The majority were early stage carcinomas (FIGO I n = 27 [64.3%], FIGO II n = 4 [9.5%], FIGO III n = 11 [26.2%]. Seven pts (16.7%) received adjuvant chemotherapy (ChT). Pts were treated with external beam radiotherapy (EBRT) and brachytherapy (IVB) boost.ResultsFive-year local recurrence free survival (LRFS), distant metastases free survival (DMFS) and overall survival (OS) were 85.4%, 78%, and 64.5% respectively. LRFS was better with lower pT stage, without lymphangiosis (L0), without haemangiosis (V0) and negative resection margins (R0). DMFS was prolonged in lymph node negatives (N0), L0, V0 and R0. OS was improved in younger pts, N0, L0, V0 and after lymphadenectomy (LNE). Multivariate analysis revealed haemangiosis (V1) as the only independent prognostic factor for OS (p = .014) and DMFS (p = .008). For LRFS pT stage remained as an independent prognostic factor (p = .028).ConclusionsAdjuvant RT with EBRT/IVB ensures adequate local control in type II EC, but control rates remain lower than in type I EC. A benefit of additional adjuvant ChT could not be demonstrated and a general omission of EBRT cannot be recommended at this point. Lymphovascular infiltration and pT stage might be the best predictive factors for a benefit from combined local and systemic treatment.

Frequent Expression of Napsin A in Clear Cell Carcinoma of the Endometrium

The histotyping of high-grade endometrial carcinomas with clear cells may be subject to significant interobserver variability, which suggests that a biomarker that can distinguish endometrial clear cell carcinoma (CCC) from its mimics would be of diagnostic utility. This study assessed the usefulness of napsin A immunohistochemistry in the diagnosis of CCC, on the basis of an analysis of 77 cases diagnosed as such at 9 institutions. After being independently reviewed by a subset of 3 pathologists, cases for which there was diagnostic consensus among all 3 reviewers in agreement with the primary contributor (n=60) were used to establish a "consensus group" that served as a gold standard relative to which napsin A performance was assessed. Duplicate, 1.0-mm-core tissue microarrays were constructed from the 54 cases in the consensus group for which requisite materials were available, as well as from 49 endometrial endometrioid carcinomas (all grades) and 17 endometrial serous carcinomas. Napsin A immunohistochemical analysis was performed on the microarrays and on the 17 cases for which there was no diagnostic consensus, with scoring based on the proportion of immunoreactive cells (0, 1+, 2+, and 3+ indicative of 0, 1% to 25%, 26% to 49%, and ≥50% immunoreactive cells, respectively). The distribution of scores for the 49 CCC cases with evaluable cores was as follows: 0, n=6; 1+, n=6; 2+, n=8; 3+, n=29. Among the evaluable cases, the frequency of ≥1+ napsin A immunoreactivity was significantly higher in CCCs (43/49, 88%) than in endometrial serous carcinomas (1/13, 7.7%; P<0.0001) and endometrial endometrioid carcinomas (0/49, 0%; P<0.0001). The sensitivity, specificity, negative predictive value, and positive predictive value of ≥1+ napsin A expression in predicting the consensus clear cell histotype were 0.88 (95% confidence interval [CI], 0.75-0.95), 0.98 (95% CI, 0.9-1), 0.91 (95% CI, 0.86-0.96), and 0.98 (95% CI, 0.86-1), respectively. Napsin A expression was not associated with survival or clinicopathologic factors. In the group of cases without diagnostic consensus for CCC, 50% showed ≥1+ napsin A expression; all napsin A-negative cases had previously been classified as non-CCC by ≥2 reviewers, whereas only 37.5% of the napsin A-positive cases had been classified as CCC by 2 of the 3 reviewers. In conclusion, napsin A is a sensitive and specific biomarker of the clear cell histotype in endometrial carcinomas and accordingly may have diagnostic utility in their histotyping.

Immunohistochemical characterization of prototypical endometrial clear cell carcinoma—diagnostic utility of HNF‐1β and oestrogen receptor

The great majority of ovarian clear cell carcinomas have a hepatocyte nuclear factor 1 homeobox B (HNF-1β)-positive and oestrogen receptor (ER)-negative immunoprofile. However, the pattern of HNF-1β and ER immunostaining in clear cell carcinomas of the endometrium and the usefulness of this panel in distinguishing clear cell carcinoma from other histological types of endometrial carcinoma have yet to be well defined. We examined the immunostaining patterns of HNF-1β, ER and p53 in 15 morphologically classic pure endometrial clear cell carcinomas, and compared these patterns with 15 endometrioid and 15 serous carcinomas of the endometrium. We observed the presence of diffuse (>70%) moderate to strong nuclear HNF-1β staining and negative ER staining in 14 of 15 clear cell carcinomas, with the remaining case showing both diffuse strong nuclear HNF-1β staining and focal ER staining. In comparison, only one of 15 serous carcinomas and none of 15 endometrioid carcinomas showed a combination of diffuse moderate to strong HNF-1β nuclear staining and negative ER staining. Aberrant p53 immunostaining was observed in five of 15 (33%) clear cell carcinomas. Overall, our findings demonstrate that, similarly to the situation for the ovary, a diagnostic panel of HNF-1β and ER may be considered for separating clear cell carcinoma from endometrioid and serous carcinoma of the endometrium.

Utility of α-methylacyl-coenzyme-A racemase (p504s) immunohistochemistry in distinguishing endometrial clear cell carcinomas from serous and endometrioid carcinomas

The expression of α-methylacyl-coenzyme-A racemase (AMACR) has previously been reported in 75% to 100% of urethral/bladder clear cell carcinomas, tumors that are known to display broad phenotypic overlap with their identically named müllerian counterparts. Herein, we assess the utility of AMACR in distinguishing endometrial clear cell carcinomas (CCCs) from endometrial serous carcinomas (ESCs) and endometrial endometrioid carcinomas (EECs). A total of 111 endometrial carcinomas in a tissue microarray, including 49 CCCs, 13 ESCs, and 49 EECs, were assessed for AMACR immunoreactivity, with results scored semiquantitatively (scores 0, 1+, 2+, 3+ for 0%, 1%-5%, 6%-50%, >50% immunoreactive cells, respectively). Fifty (45%) of the 111 carcinomas were AMACR positive, with the following score distribution: CCC: 0 (n = 12), 1+ (n = 12), 2+ (n = 3), 3+ (n = 22); EEC: 0 (n = 38), 1+ (n = 4), 2+ (n = 4), 3+ (n = 3); ESC: 0 (n = 11), 1+ (n = 1), 2+ (n = 0), 3+ (n = 1). AMACR expression was significantly more frequent in CCC (75%) than in ESC (15%) or EEC (22%); P < .0001. The sensitivity and specificity of AMACR expression in classifying a carcinoma as CCC were 0.75 (95% confidence interval [CI], 0.61-0.86) and 0.79 (95% CI, 0.66-0.88), respectively, with an odds ratio of 11.62 (95% CI, 5-28; P < .001) and an area under the curve of 0.79 (95% CI, 0.68-0.88). These findings indicate that AMACR expression is strongly associated with CCC and displays a relatively robust diagnostic test performance. However, its practical utility may be limited by the focal nature of its expression in 32% of the AMACR-positive CCC cases as well as its expression in 15% to 22% of the non-CCC histotypes.

Abstract A25: PPP2R1A mutations affect PP2A complex formation, and novel protein interactions in gynecological carcinoma cell lines

Abstract Gynecological malignancies (ovarian clear cell and endometrioid carcinomas, endometrial serous, endometrioid and clear cell carcinomas) harbor somatic mutations in the gene PPP2R1A, the scaffolding subunit of the serine/threonine protein phosphatase 2A (PP2A) complex. The PP2A complex is composed of an A (scaffold), C (catalytic) subunit, and different B (regulatory) subunits. The PPP2R1A mutations are proposed to play a role in PP2A B-subunit binding, and thus formation of a functional PP2A phosphatase complex. The aim of this study is to determine how PPP2R1A mutations affect the binding of B-subunits and other novel interactions within the context of ovarian and endometrial carcinomas. The ovarian clear cell line RMG2 and endometrial carcinoma cell lines Hec-1-A and Hec50 all harbor PPP2R1A mutations. The mismatch repair deficient cell line Hec-1-A express a PPP2R1A W257L heterozygous mutation; therefore somatic cell knockout technology was used to generate isogenic PPP2R1A clones that express only mutant PPP2R1A, and clones that express more wild-type than mutant. These Hec-1-A isogenic cell lines were used for PPP2R1A co-immunoprecipiation (Co-IP) followed by MS/MS-SWATHTM analysis to identify PP2A B-subunits and other novel protein interactions. Our preliminary results using Hec-1-A isogenic cell lines suggest that some PP2A B-subunits (PPP2R5D and PPP2R2A) are found to be decreased in abundance in the mutant cell lines. We were able to identify the catalytic subunit (PPP2CA) in PPP2R1A Co-IPs in all cell lines. We also show that PPP2R1A interacts with novel proteins such as histones (Histone 1, Histone H2B type 1-N), ubiquitin ligases (TRIM21) and proteins involved in mitotic control (ANKLE2). Of particular interest we have identified the cancer gene nucleophosmin (NPM1) as a potential novel interactor of PP2A. We have established the isogenic Hec-1-A cell lines as a model system to determine the functional effects of a somatic PPP2R1A mutation. These cell lines have shown that the PPP2R1A mutation affects the ability of some B-subunits to interact and form the PP2A complex. We have also shown that PP2A may interact with novel proteins such as NPM1 and histones. Ongoing work will be completed to determine how these interactions function in the context of ovarian and endometrial carcinomas. Citation Format: Melissa K. McConechy, Vincent Chen, Gregg Morin, James Brenton, David G. Huntsman. PPP2R1A mutations affect PP2A complex formation, and novel protein interactions in gynecological carcinoma cell lines. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: From Concept to Clinic; Sep 18-21, 2013; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2013;19(19 Suppl):Abstract nr A25.

The clinicopathologic significance of p53 and BAF-250a (ARID1A) expression in clear cell carcinoma of the endometrium

TP53 mutation (and associated p53 protein overexpression) is probably a negative prognostic marker in endometrial cancers, but its relevance in the rarer histologic subtypes, including clear cell carcinomas, has not been delineated. Preclinical studies suggest functional interactions between p53 and the BAF250a protein, the product of a tumor suppressor gene ARID1A (adenine-thymine (AT)-rich interactive domain containing protein 1A) that is frequently mutated in ovarian clear cell carcinoma. In this study, we evaluated the significance of p53 and BAF250a expression, as assessed by immunohistochemistry, in a group of 50 endometrial clear cell carcinomas. Of 50 cases, 17 (34%) were p53+, and the remaining 33 cases had a p53 wild-type (p53-wt) immunophenotype. Of the 11 relapses/recurrences in the entire data set, 73% were in the p53+ group (P=0.008). On univariate analyses, the median overall survival for the p53-wt patients (83 months) was longer than the p53+ patients (63 months) (P=0.07), and the median progression-free survival for the p53-wt group (88 months) was significantly longer than the p53+ group (56 months) (P=0.01). On multivariate analyses, p53 expression was not associated with reduced overall or progression-free survival. In addition, p53 status was not significantly associated with pathologic stage or morphologic patterns. Of the 50 cases, 10 (20%) showed a complete loss of BAF250a expression. There was no significant correlation between p53 and BAF250a expression. The p53+/BAF250a−, p53+/BAF250a+, p53-wt/BAF250a+ and p53-wt/BAF250a− composite immunophenotypes were identified in 8%, 26%, 54% and 12% of cases, respectively, and neither loss of BAF250a expression nor composite p53/BAF250a expression patterns were associated with reduced overall or progression-free survival. In conclusion, a significant subset of CCC express p53, and these cases are apparently not definable by their morphologic features. P53 expression may be a negative prognostic factor in this histotype, and warrants additional studies. Loss of BAF250a expression has no prognostic significance in endometrial clear cell carcinomas.