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1.  Pathogenesis and New Therapeutic Targets of Ovarian Cancer 
Journal of Oncology  2012;2012:867512.
doi:10.1155/2012/867512
PMCID: PMC3437294  PMID: 22969800
2.  Roles of Deletion of Arid1a, a Tumor Suppressor, in Mouse Ovarian Tumorigenesis 
The chromatin remodeling gene, ARID1A, has been implied as a tumor suppressor, and its somatic inactivating mutations occur in a wide variety of human cancers, most frequently in ovarian and uterine endometrioid and ovarian clear cell carcinomas. Tumors with ARID1A mutations also frequently harbor PTEN or PIK3CA mutations, suggesting their collaboration in tumorigenesis. Here, we used a conditional knockout mouse model in which Arid1a and Pten were deleted either individually or in combination in the mouse ovarian surface epithelium. After 6 months, 59.1% of mice with Arid1a and Pten double knockout developed ovarian endometrioid or undifferentiated carcinoma, whereas the remaining mice showed hyperplasia of ovarian surface epithelium. In contrast, 52 mice with homozygous or heterozygous deletion in either Arid1a or Pten did not develop ovarian lesions. These results demonstrate that inactivation of Arid1a alone is insufficient for tumor initiation but it requires additional genetic alteration(s) such as Pten deletion to drive tumorigenesis.
doi:10.1093/jnci/dju146
PMCID: PMC4056776  PMID: 24899687
3.  The emerging roles of ARID1A in tumor suppression 
Cancer Biology & Therapy  2014;15(6):655-664.
ARID1A has emerged as a tumor suppressor gene, which is mutated in a broad spectrum of cancers, especially in those arising from ectopic or eutopic endometrium. As a subunit of SWI/SNF chromatin remodeler, ARID1A facilitates target-specific binding of SWI/SNF complexes to chromatin, thereby altering the accessibility of chromatin to a variety of nuclear factors. In human cancer, ARID1A possesses not only features of a gatekeeper, regulating cell cycle progression, but also features of a caretaker, preventing genomic instability. An increasing body of evidence suggests crosstalk between ARID1A and PI3K/Akt pathways, and between ARID1A and p53. In this review, we discuss the spectrum of ARID1A alterations in cancers, tumor suppression mechanisms of ARID1A, oncogenic pathways cooperating with ARID1A, and clinical implications of ARID1A mutation.
doi:10.4161/cbt.28411
PMCID: PMC4049780  PMID: 24618703
ARID1A; PI3K; SWI/SNF; chromatin remodeling complex; clear cell carcinoma; endometrial cancer; endometrioid carcinoma; gastric cancer; ovarian cancer; tumor suppressor gene
4.  Rsf-1, a chromatin remodeling protein, interacts with cyclin E1 and promotes tumor development 
The Journal of pathology  2013;229(4):559-568.
Chromosome 11q13.5 containing RSF1 (HBXAP), a gene involved in chromatin remodeling, is amplified in several human cancers including ovarian carcinoma. Our previous studies demonstrated requirement of Rsf-1 for cell survival in cancer cells, which contributed to tumor progression; however, its role in tumorigenesis has not yet been elucidated. In this study, we co-immunoprecipitated proteins with Rsf-1 followed by nanoelectrospray mass spectrometry and identified cyclin E1, besides SNF2H, as one of the major Rsf-1 interacting proteins. Like RSF1, CCNE1 is frequently amplified in ovarian cancer, and both Rsf-1 and cyclin E1 were found co-upregulated in ovarian cancer tissues. Ectopic expression of Rsf-1 and cyclin E1 in non-tumorigenic TP53mut RK3E cells led to an increase in cellular proliferation and tumor formation by activating cyclin E1-associated kinase (CDK2). Tumorigenesis was not detected if either cyclin E1 or Rsf-1 was expressed, or they were expressed in a TP53wt background. Domain mapping showed that cyclin E1 interacted with the first 441 amino acids of Rsf-1. Ectopic expression of this truncated domain significantly suppressed G1/S-phase transition, cellular proliferation and tumor formation of RK3E-p53R175H/Rsf-1/cyclin E1 cells. The above findings suggest that Rsf-1 interacts and collaborates with cyclin E1 in neoplastic transformation and TP53 mutations are prerequisite for tumor-promoting functions of RSF/cyclin E1 complex.
doi:10.1002/path.4147
PMCID: PMC4353404  PMID: 23378270
Rsf-1; HBXAP; cyclin E1; chromatin remodeling; TP53mut; cancer development
5.  Precursors of ovarian cancer in the fallopian tube: Serous tubal intraepithelial carcinoma – an update 
Ovarian tumors comprise a wide variety of entities. The largest group, epithelial ovarian carcinoma, can be classified into two main groups, type I and type II tumors. Recent advances in the understanding of ovarian cancer development have resulted in the finding of ‘serous tubal intraepithelial carcinoma’, which is believed to represent the precursor lesion in high-grade serous ovarian carcinoma. In this review, lines of evidence for this are discussed and possible future implications for clinical and research settings are outlined.
doi:10.1111/jog.12550
PMCID: PMC4352308  PMID: 25330822
epithelial ovarian cancer; fallopian tube; high-grade serous carcinoma; precursor; serous tubal intraepithelial carcinoma
6.  Loss of ALDH1A1 expression is an early event in the pathogenesis of ovarian high-grade serous carcinoma 
Tumour-initiating cells are thought to share features with normal somatic stem cells. In mice, stem cells at the ovarian hilum have been shown to express the stem cell marker, aldehyde dehydrogenase isoform 1 (ALDH1A1), and are prone to malignant transformation. The potential relevance of this finding to humans has not been established. In this study, we used immunohistochemistry to assess the distribution of ALDH1A1 staining in the epithelium of human fallopian tubes, with particular reference to the transition of tubal epithelium to mesothelium (i.e. the tubal-mesothelial junction), the ovarian surface epithelium (OSE), putative precursors of ovarian high grade serous carcinoma (HGSC), namely, serous tubal intraepithelial carcinoma (STIC) and p53 signatures, and overt HGSC. Expression of ALDH1A1 was detected in both secretory and ciliated tubal epithelial cells, tubal-mesothelial junctions and OSE, but was absent in STIC and p53 signatures. Positive staining in HGSC, when present, was typically limited to rare tumour cells. In-silico analyses of the mRNA expression dataset from The Cancer Genome Atlas revealed down-regulation of ALDH1A1 transcripts in HGSC relative to normal tubal epithelium and no association between ALDH1A1 expression levels and overall survival. Our results do not support ALDH1A1 as a marker of stem cells in human fallopian tube and demonstrate that its loss of expression is an early event in the development of HGSC.
doi:10.1038/modpathol.2014.89
PMCID: PMC4344882  PMID: 25216223
STIC; p53 signature; ALDH1; stem cells; ovarian cancer; tubal-mesothelial junction
7.  Loss of ARID1A Expression Correlates with Stages of Tumor Progression in Uterine Endometrioid Carcinoma 
ARID1A is a recently identified tumor suppressor that functions in chromatin remodeling. Inactivating mutations of ARID1A and loss of its expression most frequently occur in ovarian clear cell carcinoma, ovarian endometrioid carcinoma, and uterine endometrioid carcinoma. In this study, we performed a detailed immunostaining analysis of ARID1A in 246 cases including benign endometrium and endometrioid carcinoma at different stages of progression. Special attention was paid to recording intratumoral heterogeneity of clonal loss of ARID1A immunoreactivity. All normal endometria (n= 51) and endometrial polyps (n= 14) retained ARID1A expression. Among complex atypical hyperplasia (n= 38), 16% exhibited clonal loss of ARID1A, but none showed complete loss. Among low-grade endometrioid carcinomas (n= 88), 25% exhibited complete loss and 24% exhibited clonal loss. In contrast, 44% of high-grade endometrioid carcinomas (n= 55) showed complete loss of ARID1A and 9% exhibited clonal loss. We found that 19 high-grade carcinomas also contained concurrent low-grade carcinomas. In the high-grade areas, 63% exhibited complete loss and 11% exhibited clonal loss, whereas in the low-grade areas, 37% exhibited complete loss and 42% clonal loss. In 5 of these 19 cases, progressive loss of ARID1A from retention or clonal loss to complete loss was observed between the low-grade and high-grade areas. Overall, the percentage of complete ARID1A loss increased from 0% in complex atypical hyperplasia, to 25% in low-grade endometrioid carcinoma, to 44% in high-grade endometrioid carcinoma. These findings suggest that loss of ARID1A expression, presumably due to mutation, plays an important role in tumor progression of uterine endometrioid carcinoma.
doi:10.1097/PAS.0b013e3182889dc3
PMCID: PMC3787317  PMID: 24076775
8.  Pathogenesis and the role of ARID1A mutation in endometriosis-related ovarian neoplasms 
Advances in anatomic pathology  2013;20(1):45-52.
Endometriosis-related ovarian neoplasms (ERONs) are a unique group of tumors as they are associated with endometriosis, especially endometriosis presenting as an ovarian endometriotic cyst (endometrioma). ERONs include clear cell carcinoma, endometrioid carcinoma, and seromucinous borderline tumor. A growing body of evidence from both clinicopathological and molecular studies suggests that most, if not all, ERONs develop from endometriotic cyst epithelium through different stages of tumor progression. The endometriotic cyst contains abundant iron-induced reactive oxygen species which are thought to be mutagenic, and chronic exposure of cystic epithelium to this microenvironment facilitates the accumulation of somatic mutations that ultimately result in tumor development. Molecular analyses of ERONs, including genome-wide screens, have identified several molecular genetic alterations that lead to aberrant activation or inactivation of pathways involving ARID1A, PI3K, Wnt, and PP2A. Among all molecular genetic changes identified to date, inactivating mutations of the ARID1A tumor suppressor gene are the most common in ERON. Understanding the molecular changes and pathogenesis involved in the development of ERON is fundamental for future translational studies aimed at designing new diagnostic tests for early detection and identifying critical molecular features for targeted therapeutics.
doi:10.1097/PAP.0b013e31827bc24d
PMCID: PMC3523307  PMID: 23232571
endometriosis-related ovarian neoplasms; clear cell carcinoma; endometrioid carcinoma; endometrial carcinoma; ARID1A
9.  The diagnostic and biological implications of laminin expression in serous tubal intraepithelial carcinoma 
There is compelling evidence to suggest that serous tubal intraepithelial carcinoma (STIC) is the likely primary site for the development of pelvic high-grade serous carcinomas (HGSCs). Identifying molecules that are upregulated in STIC is important not only to provide biomarkers to assist in the diagnosis of STIC but also to elucidate our understanding of the pathogenesis of HGSC. In this study, we performed RNA sequencing to compare transcriptomes between HGSC and normal fallopian tube epithelium (FTE), and identified LAMC1 encoding laminin γ1 as one of the preferentially upregulated gene associated with HGSC. RT-PCR further validated LAMC1 upregulation in HGSC as compared to normal FTE. Immunohistochemistry was performed on 32 cases of concurrent HGSC and STIC. The latter was diagnosed based on morphology, TP53 mutations, p53 and Ki-67 immunohistochemical pattern. Laminin γ1 immunostaining intensity was found to be significantly higher in STIC and HGSC compared to adjacent FTE in all cases (p< 0.001). In normal FTE, laminin γ1 immunoreactivity was predominantly localized in the basement membrane or on the apical surface of ciliated cells whereas in STIC and HGSC cells, laminin γ1 staining was diffuse and intense throughout the cytoplasm. More importantly, strong laminin γ1 staining was detected in all 13 STICs which lacked p53 immunoreactivity due to null mutations. These findings suggest that the overexpression of laminin γ1 immunoreactivity and alteration of its staining pattern in STICs can serve as a useful tissue biomarker, especially for those STICs that are negative for p53 and have a low Ki-67 labeling index.
doi:10.1097/PAS.0b013e31825ec07a
PMCID: PMC3500426  PMID: 22892598
ovarian cancer; serous tubal intraepithelial carcinoma; STIC; laminin; LAMC1
10.  Telomere length in different histologic types of ovarian carcinoma with emphasis on clear cell carcinoma 
Ovarian carcinoma is composed of a heterogeneous group of tumors with distinct clinico-pathological and molecular features. Alteration of telomerase activity has been reported in ovarian tumors but the pattern of telomere length in their specific histological subtypes has not been reported. In this study, we performed quantitative telomere fluorescence in situ hybridization on a total of 219 ovarian carcinomas including 106 high-grade serous carcinomas, 26 low-grade serous carcinomas, 56 clear cell carcinomas and 31 low-grade endometrioid carcinomas. The mean relative telomere length of carcinoma to stromal cells was calculated as a telomere index. This index was significantly higher in clear cell carcinoma compared with the other histologic types (P= 0.007). Overall there was no association between the telomere index and mortality, but when stratified by histologic types, the hazard ratio for death among women with clear cell carcinoma with a telomere index >1 was significantly increased at 4.93 (95% CI 1.64–14.86, P= 0.005) when compared with those with a telomere index ≤1. In conclusion, our results provide new evidence that telomere length significantly differs by histologic type in ovarian carcinoma. Specifically, clear cell carcinomas have longer mean relative telomere lengths compared with the other histologic types and longer telomeres in clear cell carcinoma are associated with increased mortality suggesting that aberrations in telomere length may have an important role in the development and progression of this neoplasm.
doi:10.1038/modpathol.2011.67
PMCID: PMC4763925  PMID: 21499239
clear cell carcinoma; FISH; telomere; ovarian cancer
11.  Identification of Molecular Pathway Aberrations in Uterine Serous Carcinoma by Genome-wide Analyses 
Background
Uterine cancer is the fourth most common malignancy in women, and uterine serous carcinoma is the most aggressive subtype. However, the molecular pathogenesis of uterine serous carcinoma is largely unknown. We analyzed the genomes of uterine serous carcinoma samples to better understand the molecular genetic characteristics of this cancer.
Methods
Whole-exome sequencing was performed on 10 uterine serous carcinomas and the matched normal blood or tissue samples. Somatically acquired sequence mutations were further verified by Sanger sequencing. The most frequent molecular genetic changes were further validated by Sanger sequencing in 66 additional uterine serous carcinomas and in nine serous endometrial intraepithelial carcinomas (the preinvasive precursor of uterine serous carcinoma) that were isolated by laser capture microdissection. In addition, gene copy number was characterized by single-nucleotide polymorphism (SNP) arrays in 23 uterine serous carcinomas, including 10 that were subjected to whole-exome sequencing.
Results
We found frequent somatic mutations in TP53 (81.6%), PIK3CA (23.7%), FBXW7 (19.7%), and PPP2R1A (18.4%) among the 76 uterine serous carcinomas examined. All nine serous carcinomas that had an associated serous endometrial intraepithelial carcinoma had concordant PIK3CA, PPP2R1A, and TP53 mutation status between uterine serous carcinoma and the concurrent serous endometrial intraepithelial carcinoma component. DNA copy number analysis revealed frequent genomic amplification of the CCNE1 locus (which encodes cyclin E, a known substrate of FBXW7) and deletion of the FBXW7 locus. Among 23 uterine serous carcinomas that were subjected to SNP array analysis, seven tumors with FBXW7 mutations (four tumors with point mutations, three tumors with hemizygous deletions) did not have CCNE1 amplification, and 13 (57%) tumors had either a molecular genetic alteration in FBXW7 or CCNE1 amplification. Nearly half of these uterine serous carcinomas (48%) harbored PIK3CA mutation and/or PIK3CA amplification.
Conclusion
Molecular genetic aberrations involving the p53, cyclin E–FBXW7, and PI3K pathways represent major mechanisms in the development of uterine serous carcinoma.
doi:10.1093/jnci/djs345
PMCID: PMC3692380  PMID: 22923510
12.  Loss of ARID1A expression is an early molecular event in tumor progression from ovarian endometriotic cyst to clear cell and endometrioid carcinoma 
OBJECTIVES
ARID1A is a recently identified tumor suppressor participating in chromatin remodeling. Somatic inactivating mutations of ARID1A and loss of its expression occur most frequently in ovarian clear cell and endometrioid carcinomas and uterine endometrioid carcinomas. Since endometriosis is thought to be a precursor of most ovarian clear cell and endometrioid carcinomas, we undertook an analysis of ARID1A expression of these tumors arising within an endometriotic cyst (endometrioma).
MATERIALS/METHODS
Our immunohistochemical study set consisted of 47 endometriotic cysts containing clear cell carcinoma in 24 cases, well-differentiated ovarian endometrioid carcinoma in 20 and mixed clear cell and endometrioid carcinoma in 3.
RESULTS
ARID1A loss was observed in 31 (66%) of 47 carcinomas and therefore these cases were informative for determining the temporal sequence of loss of ARID1A expression in tumor progression. In 16 of the 47 cases, ARID1A immunoreactivity was retained in both the endometriotic cyst and the carcinoma and thus these cases were not informative. All of the 31 informative cases showed loss of ARID1A immunoreactivity in the carcinoma and in the endometriotic cyst epithelium in direct continuity with the carcinoma but not in the cyst epithelium that was not adjacent to the tumor.
CONCLUSIONS
The findings in this study provide cogent evidence that loss of ARID1A function as shown by loss of expression, presumably due to mutations, is an early molecular event, occurring before malignant transformation, in the development of the majority of ovarian clear cell and endometrioid carcinomas arising in endometriomas.
doi:10.1097/IGC.0b013e31826b5dcc
PMCID: PMC3460070  PMID: 22976498
13.  NAC1 is an actin-binding protein that is essential for effective cytokinesis in cancer cells 
Cancer research  2012;72(16):4085-4096.
NAC1 is a transcriptional co-repressor protein that is essential to sustain cancer cell proliferation and migration. However, the underlying molecular mechanisms of NAC1 function in cancer cells remain unknown. In this study, we show that NAC1 functions as an actin monomer binding protein. The conserved BTB protein interaction domain in NAC1 is the minimal region for actin binding. Disrupting NAC1 complex function by dominant negative or siRNA strategies reduced cell retraction and abscission during late stage cytokinesis, causing multinucleation in cancer cells. In Nac1-deficient murine fibroblasts, restoring NAC1 expression was sufficient to partially avert multinucleation. We found that siRNA-mediated silencing of the actin binding protein profilin-1 in cancer cells caused a similar multinucleation phenotype and that NAC1 modulated the binding of actin to profillin-1. Taken together, our results indicate that the NAC1/actin/profilin-1 complex is crucial for cancer cell cytokinesis, with a variety of important biological and clinical implications.
doi:10.1158/0008-5472.CAN-12-0302
PMCID: PMC3421062  PMID: 22761335
actin; cytokinesis; NAC1; profilin-1
14.  Clonality analysis of combined Brenner and mucinous tumours of the ovary reveals their monoclonal origin 
The Journal of pathology  2015;237(2):146-151.
The derivation of ovarian intestinal-type mucinous tumours is not well established. Some are derived from teratomas but the origin of the majority is not clear. It has been recently proposed that the non-germ cell group may be derived from Brenner tumours, as the association of a mucinous tumour with a Brenner tumour is frequently observed. In order to explore the histogenesis of these neoplasms, we undertook a clonality analysis of the two components of ten combined Brenner and mucinous tumours using a human androgen receptor gene (HUMARA) assay. All eight informative cases of ten showed a concordant X-chromosome inactivation pattern between the two tumour components, indicative of a shared clonal origin (p = 0.0039). Microsatellite genotyping in five of the combined tumours displayed an identical heterozygous pattern with paired Fallopian tube tissue, indicative of a somatic cell origin. In addition, paired box protein 8, a highly sensitive Müllerian epithelial marker, was not detected by immunohistochemistry in either tumour component in any of the ten tumours, suggesting that this subset of mucinous tumours does not originate from Müllerian-derived epithelium. In conclusion, this study demonstrates that in combined mucinous and Brenner tumours, there is a shared clonal relationship between the two different tumour components and suggests that some pure mucinous tumours may develop from a Brenner tumour in which the Brenner tumour component becomes compressed and obliterated by an expanding mucinous neoplasm.
doi:10.1002/path.4572
PMCID: PMC4703556  PMID: 26095692
ovary; Brenner tumour; mucinous tumour; histogenesis; X-chromosome inactivation; HUMARA; clonality analysis
15.  Ki-67 Labeling Index as an Adjunct in the Diagnosis of Serous Tubal Intraepithelial Carcinoma 
Summary
There is mounting evidence that serous tubal intraepithelial carcinoma (STIC) may be the immediate precursor of ovarian high-grade serous carcinoma (HGSC) but the criteria for its diagnosis are not well established as highlighted in a recent study showing that interobserver reproducibility, even among expert gynecologic pathologists, was moderate at best. Given the clinical significance of a diagnosis of STIC in a patient who has no other evidence of ovarian carcinoma, this is a serious issue that we felt needed to be addressed. Although it is not clear, at this time, whether such a patient should or should not be treated, the importance of an accurate and reproducible diagnosis of precursors of ovarian carcinoma cannot be underestimated. We hypothesized that an elevated Ki-67 labeling index may aid the diagnosis of STIC. Accordingly, we compared the Ki-67 index of STIC and HGSC to normal fallopian tube epithelium (FTE) in the same patients and to a control group of patients without carcinoma, matched for age. A total of 41 STICs were analyzed, of which 35 were associated with a concurrent HGSC. In FTE, immunoreactivity for Ki-67 was restricted to a few scattered cells (mean 2.0%). No statistically significant difference was found between patients with and without HGSC (P>0.05). However, both STICs and HGSC had significantly higher Ki-67 indices than normal FTE (P<0.0001). STICs uniformly had an elevated Ki-67 labeling index that ranged from 11.7% to 71.1% (average 35.6%). There was no correlation of the Ki-67 labeling index in the STICs and the associated HGSC, as the labeling index was lower in STIC in 18/35 (51.4%) whereas it was higher in 17/35 (48.6%) (P=0.86). In conclusion, the findings in this study indicate that compared with FTE, STICs have a significantly higher Ki-67 index similar to HGSC. Accordingly, the Ki-67 index can aid the diagnosis of intraepithelial tubal proliferations suspicious for STIC. Therefore, we propose that a Ki-67 index of 10% is a useful diagnostic tool to distinguish STICs from normal FTE.
doi:10.1097/PGP.0b013e31824cbeb4
PMCID: PMC3715095  PMID: 22833080
Ovarian cancer; Serous tubal intraepithelial carcinoma; Ki-67; Serous; STIC
16.  KDDN: an open-source Cytoscape app for constructing differential dependency networks with significant rewiring 
Bioinformatics  2014;31(2):287-289.
Summary: We have developed an integrated molecular network learning method, within a well-grounded mathematical framework, to construct differential dependency networks with significant rewiring. This knowledge-fused differential dependency networks (KDDN) method, implemented as a Java Cytoscape app, can be used to optimally integrate prior biological knowledge with measured data to simultaneously construct both common and differential networks, to quantitatively assign model parameters and significant rewiring p-values and to provide user-friendly graphical results. The KDDN algorithm is computationally efficient and provides users with parallel computing capability using ubiquitous multi-core machines. We demonstrate the performance of KDDN on various simulations and real gene expression datasets, and further compare the results with those obtained by the most relevant peer methods. The acquired biologically plausible results provide new insights into network rewiring as a mechanistic principle and illustrate KDDN’s ability to detect them efficiently and correctly. Although the principal application here involves microarray gene expressions, our methodology can be readily applied to other types of quantitative molecular profiling data.
Availability: Source code and compiled package are freely available for download at http://apps.cytoscape.org/apps/kddn
Contact: yuewang@vt.edu
Supplementary information: Supplementary data are available at Bioinformatics online.
doi:10.1093/bioinformatics/btu632
PMCID: PMC4287948  PMID: 25273109
17.  GATA-3 EXPRESSION IN TROPHOBLASTIC TISSUES: An Immunohistochemical Study of 445 Cases, Including Diagnostic Utility 
Immunohistochemical expression of GATA-3 is seen predominantly in non-neoplastic bladder and breast epithelium and their respective carcinomas; however, data on expression in normal and lesional trophoblastic tissues are limited. Immunohistochemical staining for GATA-3 was assessed in a range of normal/lesional trophoblastic tissues and tumors in the differential diagnosis (n=445), including non-molar products of conceptions/2nd and 3rd trimester placentas/ ectopic pregnancies, hydatidiform moles, placental site nodules, normal/exaggerated implantation sites, choriocarcinomas, epithelioid trophoblastic tumors, placental site trophoblastic tumors, atypical smooth muscle tumors (including leiomyosarcoma), and cervical and pulmonary squamous cell carcinomas. The extent of expression (0 to 4+) and intensity (weak to strong) were recorded. All cases with developing trophoblast/non-neoplastic trophoblastic proliferations and 81% of trophoblastic neoplasms were positive. Of all non-neoplastic trophoblast cell types, expression was observed in cytotrophoblast in 89% of cases, syncytiotrophoblast in 50%, intermediate trophoblast in 100%, and villous trophoblastic columns in 100%. Increasing gestational age was associated with a decrease in extent/intensity of expression in non-neoplastic cytotrophoblast and syncytiotrophoblast while intermediate trophoblast maintained diffuse and strong expression from early to late gestation (p<0.0001). Eighty-nine percent of normal/exaggerated implantation sites showed 3+ or 4+ expression while staining in 55% of placental site nodules was 1+ or 2+. Staining for GATA-3 was present in 78% of choriocarcinomas, 95% of epithelioid trophoblastic tumors, and 71% of placental site trophoblastic tumors. While the number of choriocarcinomas and placental site trophoblastic tumors that showed a spectrum of expression ranging from negative to diffuse was relatively evenly distributed, 81% of epithelioid trophoblastic tumors had 3+ or 4+ staining. None of the atypical smooth muscle tumors and 3% of squamous cell carcinomas were positive, all of which exhibited weak staining. We conclude that GATA-3 is frequently expressed in normal and lesional trophoblastic tissues. It is also differentially expressed in intermediate trophoblast and cytotrophoblast/syncytiotrophoblast, which varies according to time during pregnancy. This study expands the spectrum of neoplasms known to express GATA-3. Thus, recognition of expression in trophoblastic tumors is important because it can present a diagnostic pitfall in the assessment of suspected metastatic bladder or breast carcinomas involving the gynecologic tract. In the evaluation of diagnostically problematic tumors for which trophoblastic neoplasms are in the differential diagnosis, such as leiomyosarcoma and squamous cell carcinoma, GATA-3 can be included as part of an immunohistochemical panel particularly when other trophoblastic markers are either not available or yield ambiguous results.
doi:10.1097/PAS.0000000000000315
PMCID: PMC4268033  PMID: 25188865
GATA-3; trophoblast; choriocarcinoma; leiomyosarcoma; squamous cell carcinoma
18.  UNDO: a Bioconductor R package for unsupervised deconvolution of mixed gene expressions in tumor samples 
Bioinformatics  2014;31(1):137-139.
Summary: We develop a novel unsupervised deconvolution method, within a well-grounded mathematical framework, to dissect mixed gene expressions in heterogeneous tumor samples. We implement an R package, UNsupervised DecOnvolution (UNDO), that can be used to automatically detect cell-specific marker genes (MGs) located on the scatter radii of mixed gene expressions, estimate cellular proportions in each sample and deconvolute mixed expressions into cell-specific expression profiles. We demonstrate the performance of UNDO over a wide range of tumor–stroma mixing proportions, validate UNDO on various biologically mixed benchmark gene expression datasets and further estimate tumor purity in TCGA/CPTAC datasets. The highly accurate deconvolution results obtained suggest not only the existence of cell-specific MGs but also UNDO’s ability to detect them blindly and correctly. Although the principal application here involves microarray gene expressions, our methodology can be readily applied to other types of quantitative molecular profiling data.
Availability and implementation: UNDO is available at http://bioconductor.org/packages.
Contact: yuewang@vt.edu
Supplementary information: Supplementary data are available at Bioinformatics online.
doi:10.1093/bioinformatics/btu607
PMCID: PMC4271149  PMID: 25212756
19.  Molecular Alterations of TP53 are a Defining Feature of Ovarian High-Grade Serous Carcinoma: A Rereview of Cases Lacking TP53 Mutations in The Cancer Genome Atlas Ovarian Study 
Summary
The Cancer Genome Atlas has reported that 96% of ovarian high-grade serous carcinomas (HGSCs) have TP53 somatic mutations suggesting that mutation of this gene is a defining feature of this neoplasm. In the current study, 5 gynecologic pathologists independently evaluated hematoxylin and eosin slides of 14 available cases from The Cancer Genome Atlas classified as HGSC that lacked a TP53 mutation. The histologic diagnoses rendered by these pathologists and the accompanying molecular genetic data are the subject of this report. Only 1 case (Case 5), which contained a homozygous deletion of TP53, had unanimous interobserver agreement for a diagnosis of pure HGSC. In 1 case (Case 3), all 5 observers (100%) rendered a diagnosis of HGSC; however, 3 observers (60%) noted that the histologic features were not classic for HGSC and suggested this case may have arisen from a low-grade serous carcinoma (arisen from an alternate pathway compared with the usual HGSC). In 2 cases (Cases 4 and 12), only 3 observers (60%) in each case, respectively, interpreted it as having a component of HGSC. In the remaining 10 (71%) of tumors (Cases 1, 2, 6–11, 13, and 14), the consensus diagnosis was not HGSC, with individual diagnoses including low-grade serous carcinoma, high-grade endometrioid carcinoma, HGSC, metastatic carcinoma, clear cell carcinoma, atypical proliferative (borderline) serous tumor, and adenocarcinoma, not otherwise specified. Therefore, 13 (93%) of the tumors (Cases 1–4 and 6–14) were either not a pure HGSC or represented a diagnosis other than HGSC, all with molecular results not characteristic of HGSC. Accordingly, our review of the TP53 wild-type HGSCs reported in The Cancer Genome Atlas suggests that 100% of de novo HGSCs contain TP53 somatic mutations or deletions, with the exception of the rare HGSCs that develop from a low-grade serous tumor precursor. We, therefore, propose that lack of molecular alterations of TP53 are essentially inconsistent with the diagnosis of ovarian HGSC and that tumors diagnosed as such should be rigorously reassessed to achieve correct classification.
doi:10.1097/PGP.0000000000000207
PMCID: PMC4696053  PMID: 26166714
The Cancer Genome Atlas; TCGA; TP53; High-grade serous carcinoma
20.  Immunohistochemical expression of ARID1A in penile squamous cell carcinomas: a tissue microarray study of 112 cases☆ 
Human pathology  2015;46(5):761-766.
Summary
ARID1A, a member of the chromatin remodeling genes family, has been suggested as a novel tumor suppressor gene in gynecologic malignancies. However, its role in penile cancer has yet to be determined. This study assesses the immunohistochemical expression of ARID1A in penile squamous cell carcinoma (SCC) and its association with pathologic features, human papillomavirus (HPV) status, and previously reported mammalian target of rapamycin pathway markers in the same cohort. Four tissue microarrays were constructed from 112 cases of formalin-fixed, paraffin-embedded penile SCC from Paraguay. Each tumor was sampled 3 to 12 times. ARID1A expression was evaluated by immunohistochemistry using a polyclonal rabbit anti-ARID1A (BAF250A) antibody. An H score was calculated in each spot as the sum of expression intensity (0-3+) by extent (0%-100%). Median H score per case was used for statistical analysis. ARID1A expression was observed in all cases, ranging from 3% to 100% of tumor cells (median, 95%). In 96 cases (86%), ARID1A expression was observed in 90% or more tumor cells. HPV DNA was detected in 20 (38%) of 52 analyzed samples. There was a significant trend of association between ARID1A and histologic grade. ARID1A expression was not associated with histologic subtype (P = .61) or HPV status (P = .18). ARID1A expression decreased with decreasing levels of PTEN expression (P = .01). ARID1A was expressed in penile SCC, in most cases at high levels. A significant trend of association was found between histologic grade and ARID1A expression, with lower ARID1A expression, lower histologic grades, and decreased PTEN expression.
doi:10.1016/j.humpath.2015.01.018
PMCID: PMC4696050  PMID: 25776029
ARID1A; BAF250A; SWI//SNF; mTOR pathway; Penile SCC
21.  Low-grade serous carcinomas of the ovary contain very few point mutations 
The Journal of pathology  2011;226(3):413-420.
It has been well established that ovarian low-grade and high-grade serous carcinomas are fundamentally different types of tumours. While the molecular genetic features of ovarian high-grade serous carcinomas are now well known, the pathogenesis of low-grade serous carcinomas, apart from the recognition of frequent somatic mutations involving KRAS and BRAF, is largely unknown. In order to comprehensively analyse somatic mutations in low-grade serous carcinomas, we applied exome sequencing to the DNA of eight samples of affinity-purified, low-grade, serous carcinomas. A remarkably small number of mutations were identified in seven of these tumours: a total of 70 somatic mutations in 64 genes. The eighth case displayed mixed serous and endometrioid features and a mutator phenotype with 783 somatic mutations, including a nonsense mutation in the mismatch repair gene, MSH2. We validated representative mutations in an additional nine low-grade serous carcinomas and 10 serous borderline tumours, the precursors of ovarian low-grade, serous carcinomas. Overall, the genes showing the most frequent mutations were BRAF and KRAS, occurring in 10 (38%) and 5 (19%) of 27 low-grade tumours, respectively. Except for a single case with a PIK3CA mutation, other mutations identified in the discovery set were not detected in the validation set of specimens. Our mutational analysis demonstrates that point mutations are much less common in low-grade serous tumours of the ovary than in other adult tumours, a finding with interesting scientific and clinical implications.
doi:10.1002/path.3967
PMCID: PMC3503448  PMID: 22102435
ovarian cancer; exome sequencing; BRAF; KRAS; somatic mutations
22.  Endocervical-type Mucinous Borderline Tumors are Related to Endometrioid Tumors Based on Mutation and Loss of Expression of ARID1A 
Summary
Nongastrointestinal-type mucinous borderline tumors have been described as displaying endocervical and serous differentiation and hence have been termed “endocervical-type” mucinous borderline tumors, “mixed epithelial papillary cystadenoma of borderline malignancy of mullerian type” or “atypical proliferative seromucinous tumors”. A striking feature of these tumors is their frequent association with endometriosis, which has been reported in a third to a half of cases. This is an unusual finding as pure endocervical and serous tumors are not usually associated with endometriosis. ARID1A is a recently identified tumor suppressor, which frequently loses its expression and is mutated in endometrium-related carcinomas including ovarian clear cell, ovarian endometrioid and uterine endometrioid carcinomas. Although ARID1A mutations and expression have been studied in gynecological cancer, the expression pattern of ARID1A has not been investigated in ovarian atypical proliferative (borderline) tumors. In this study, we analyzed ARID1A expression in serous, gastrointestinal-type and endocervical-type (seromucinous) mucinous, and endometrioid atypical proliferative (borderline) tumors using immunohistochemistry and performed mutational analysis in selected cases. We observed loss of ARID1A staining in 8 (33%) of 24 seromucinous tumors. In contrast, ARID1A staining was retained in all the other 32 tumors except in one endometrioid tumor (p<0.01). Mutational analysis was performed on two representative seromucinous tumors, which showed complete loss of ARID1A. Both tumors harbored somatic inactivating ARID1A mutations. Previous studies have reported loss of expression and/or mutation of ARID1A in 30–57% of endometrioid and clear cell carcinomas but only rarely in serous tumors. The findings in this study, showing a significantly higher frequency of loss of ARID1A expression in endocervical-type (seromucinous) tumors, presumably due to mutation, compared to the other histologic types suggest that they are molecularly related to endometrioid and clear cell tumors.
doi:10.1097/PGP.0b013e31823f8482
PMCID: PMC3501990  PMID: 22653341
ARID1A; ovarian borderline tumor
23.  ARID1A, a factor that promotes formation of SWI/SNF-mediated chromatin remodeling, is a tumor suppressor in gynecologic cancers 
Cancer research  2011;71(21):6718-6727.
ARID1A (BAF250A) promotes the formation of SWI/SNF chromatin remodeling complexes containing BRG1 or BRM. ARID1A has emerged as a candidate tumor suppressor based on its frequent mutations in ovarian clear cell and endometrioid cancers and in uterine endometrioid carcinomas. Here we report that restoring wild-type ARID1A expression in ovarian cancer cells that harbor ARID1A mutations is sufficient to suppress cell proliferation and tumor growth in mice, whereas RNAi-mediated silencing of ARID1A in non-transformed epithelial cells is sufficient to enhance cellular proliferation and tumorigenicity. Gene expression analysis identified several downstream targets of ARID1A including CDKN1A and SMAD3, which are well known p53 target genes. In support of the likelihood that p53 mediates the effects of ARID1A on these genes, we demonstrated that p53 was required and sufficient for their regulation by ARID1A. Further, we showed that CDKN1A (encoding p21) acted in part to mediate growth suppression by ARID1A. Lastly, we obtained evidence that the ARID1A/BRG1 complex interacts directly with p53 and that mutations in the ARID1A and TP53 genes were mutually exclusive in tumor specimens. Our results provide functional evidence in support of the hypothesis that ARID1A is a bona fide tumor suppressor that collaborates with p53 to regulate CDKN1A and SMAD3 transcription and tumor growth in gynecological cancers.
doi:10.1158/0008-5472.CAN-11-1562
PMCID: PMC3206175  PMID: 21900401
24.  Functional Analysis of In-frame Indel ARID1A Mutations Reveals New Regulatory Mechanisms of Its Tumor Suppressor Functions1 
Neoplasia (New York, N.Y.)  2012;14(10):986-993.
AT-rich interactive domain 1A (ARID1A) has emerged as a new tumor suppressor in which frequent somatic mutations have been identified in several types of human cancers. Although most ARID1A somatic mutations are frame-shift or nonsense mutations that contribute to mRNA decay and loss of protein expression, 5% of ARID1A mutations are in-frame insertions or deletions (indels) that involve only a small stretch of peptides. Naturally occurring in-frame indel mutations provide unique and useful models to explore the biology and regulatory role of ARID1A. In this study, we analyzed indel mutations identified in gynecological cancers to determine how these mutations affect the tumor suppressor function of ARID1A. Our results demonstrate that all in-frame mutants analyzed lost their ability to inhibit cellular proliferation or activate transcription of CDKN1A, which encodes p21, a downstream effector of ARID1A. We also showed that ARID1A is a nucleocytoplasmic protein whose stability depends on its subcellular localization. Nuclear ARID1A is less stable than cytoplasmic ARID1A because ARID1A is rapidly degraded by the ubiquitin-proteasome system in the nucleus. In-frame deletions affecting the consensus nuclear export signal reduce steady-state protein levels of ARID1A. This defect in nuclear exportation leads to nuclear retention and subsequent degradation. Our findings delineate a mechanism underlying the regulation of ARID1A subcellular distribution and protein stability and suggest that targeting the nuclear ubiquitin-proteasome system can increase the amount of the ARID1A protein in the nucleus and restore its tumor suppressor functions.
PMCID: PMC3479842  PMID: 23097632
25.  NAC1 Modulates Sensitivity of Ovarian Cancer Cells to Cisplatin via Altering the HMGB1-Mediated Autophagic Response* 
Oncogene  2011;31(8):1055-1064.
Nucleus accumbens-1 (NAC1), a nuclear factor belonging to the BTB/POZ gene family, is known to play important roles in proliferation and growth of tumor cells and in chemotherapy resistance. Yet, the mechanisms underlying how NAC1 contributes to drug resistance remain largely unclear. We reported here that autophagy was involved in NAC1-mediated resistance to cisplatin, a commonly used chemotherapeutic drug in the treatment of ovarian cancer. We found that treatment with cisplatin caused an activation of autophagy in ovarian cancer cell lines, A2780, OVCAR3, and SKOV3. We further demonstrated that knockdown of NAC1 by RNAi or inactivation of NAC1 by inducing the expression of a NAC1 deletion mutant that contains only the BTB/POZ domain significantly inhibited the cisplatin-induced autophagy, resulting in increased cisplatin cytotoxicity. Moreover, inhibition of autophagy and sensitization to cisplatin by NAC1 knockdown or inactivation were accompanied by induction of apoptosis. To confirm that the sensitizing effect of NAC1 inhibition on the cytotoxicity of cisplatin was attributed to suppression of autophagy, we assessed the effects of the autophagy inhibitors, 3-MA and chloroquine, and siRNAs targeting beclin 1 or Atg5, on the cytotoxicity of cisplatin. Treatment with 3-MA, chloroquine or beclin 1 and Atg5-targeted siRNA also enhanced the sensitivity of SKOV3, A2780 and OVCAR3 cells to cisplatin, indicating that suppression of autophagy indeed renders tumor cells more sensitive to cisplatin. Regulation of autophagy by NAC1 was mediated via high mobility group box1 (HMGB1), as the functional status of NAC1 was associated with the expression, translocation and release of HMGB1. The results of our study not only revealed a new mechanism determining cisplatin sensitivity, but also identified NAC1 as a novel regulator of autophagy. Thus, the NAC1- mediated autophagy may be exploited as a new target for enhancing the efficacy of cisplatin against ovarian cancer and other types of malignancies.
doi:10.1038/onc.2011.290
PMCID: PMC3275651  PMID: 21743489
NAC1; autophagy; apoptosis; HMGB1; cisplatin; ovarian cancer

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