Multiomic analysis of uterine leiomyomas in self-described Black and White women: molecular insights into health disparities.

BACKGROUND: Black women are at an increased risk of developing uterine leiomyomas and experiencing worse disease prognosis than White women. Epidemiologic and molecular factors have been identified as underlying these disparities, but there remains a paucity of deep, multiomic analysis investigating molecular differences in uterine leiomyomas from Black and White patients. OBJECTIVE: To identify molecular alterations within uterine leiomyoma tissues correlating with patient race by multiomic analyses of uterine leiomyomas collected from cohorts of Black and White women. STUDY DESIGN: We performed multiomic analysis of uterine leiomyomas from Black (42) and White (47) women undergoing hysterectomy for symptomatic uterine leiomyomata. In addition, our analysis included the application of orthogonal methods to evaluate fibroid biomechanical properties, such as second harmonic generation microscopy, uniaxial compression testing, and shear-wave ultrasonography analyses. RESULTS: We found a greater proportion of MED12 mutant uterine leiomyomas from Black women (>35% increase; Mann-Whitney U, P<.001). MED12 mutant tumors exhibited an elevated abundance of extracellular matrix proteins, including several collagen isoforms, involved in the regulation of the core matrisome. Histologic analysis of tissue fibrosis using trichrome staining and secondary harmonic generation microscopy confirmed that MED12 mutant tumors are more fibrotic than MED12 wild-type tumors. Using shear-wave ultrasonography in a prospectively collected cohort, Black patients had fibroids that were firmer than White patients, even when similar in size. In addition, these analyses uncovered ancestry-linked expression quantitative trait loci with altered allele frequencies in African and European populations correlating with differential abundance of several proteins in uterine leiomyomas independently of MED12 mutation status, including tetracoidpeptide repeat protein 38. CONCLUSION: Our study shows that Black women have a higher prevalence of uterine leiomyomas harboring mutations in MED12 and that this mutational status correlates with increased tissue fibrosis compared with wild-type uterine leiomyomas. Our study provides insights into molecular alterations correlating with racial disparities in uterine leiomyomas and improves our understanding of the molecular etiology underlying uterine leiomyoma development within these populations.

ProteoMixture: A cell type deconvolution tool for bulk tissue proteomic data.

Numerous multi-omic investigations of cancer tissue have documented varying and poor pairwise transcript:protein quantitative correlations, and most deconvolution tools aiming to predict cell type proportions (cell admixture) have been developed and credentialed using transcript-level data alone. To estimate cell admixture using protein abundance data, we analyzed proteome and transcriptome data generated from contrived admixtures of tumor, stroma, and immune cell models or those selectively harvested from the tissue microenvironment by laser microdissection from high grade serous ovarian cancer (HGSOC) tumors. Co-quantified transcripts and proteins performed similarly to estimate stroma and immune cell admixture (r ≥ 0.63) in two commonly used deconvolution algorithms, ESTIMATE or ConsensusTME. We further developed and optimized protein-based signatures estimating cell admixture proportions and benchmarked these using bulk tumor proteomic data from over 150 patients with HGSOC. The optimized protein signatures supporting cell type proportion estimates from bulk tissue proteomic data are available at https://lmdomics.org/ProteoMixture/.

Proteogenomic analysis of enriched HGSOC tumor epithelium identifies prognostic signatures and therapeutic vulnerabilities.

We performed a deep proteogenomic analysis of bulk tumor and laser microdissection enriched tumor cell populations from high-grade serous ovarian cancer (HGSOC) tissue specimens spanning a broad spectrum of purity. We identified patients with longer progression-free survival had increased immune-related signatures and validated proteins correlating with tumor-infiltrating lymphocytes in 65 tumors from an independent cohort of HGSOC patients, as well as with overall survival in an additional 126 HGSOC patient cohort. We identified that homologous recombination deficient (HRD) tumors are enriched in pathways associated with metabolism and oxidative phosphorylation that we validated in independent patient cohorts. We further identified that polycomb complex protein BMI-1 is elevated in HR proficient (HRP) tumors, that elevated BMI-1 correlates with poor overall survival in HRP but not HRD HGSOC patients, and that HRP HGSOC cells are uniquely sensitive to BMI-1 inhibition.

Mapping three-dimensional intratumor proteomic heterogeneity in uterine serous carcinoma by multiregion microsampling.

BACKGROUND: Although uterine serous carcinoma (USC) represents a small proportion of all uterine cancer cases, patients with this aggressive subtype typically have high rates of chemotherapy resistance and disease recurrence that collectively result in a disproportionately high death rate. The goal of this study was to provide a deeper view of the tumor microenvironment of this poorly characterized uterine cancer variant through multi-region microsampling and quantitative proteomics. METHODS: Tumor epithelium, tumor-involved stroma, and whole "bulk" tissue were harvested by laser microdissection (LMD) from spatially resolved levels from nine USC patient tumor specimens and underwent proteomic analysis by mass spectrometry and reverse phase protein arrays, as well as transcriptomic analysis by RNA-sequencing for one patient's tumor. RESULTS: LMD enriched cell subpopulations demonstrated varying degrees of relatedness, indicating substantial intratumor heterogeneity emphasizing the necessity for enrichment of cellular subpopulations prior to molecular analysis. Known prognostic biomarkers were quantified with stable levels in both LMD enriched tumor and stroma, which were shown to be highly variable in bulk tissue. These USC data were further used in a comparative analysis with a data generated from another serous gynecologic malignancy, high grade serous ovarian carcinoma, and have been added to our publicly available data analysis tool, the Heterogeneity Analysis Portal ( https://lmdomics.org/ ). CONCLUSIONS: Here we identified extensive three-dimensional heterogeneity within the USC tumor microenvironment, with disease-relevant biomarkers present in both the tumor and the stroma. These data underscore the critical need for upfront enrichment of cellular subpopulations from tissue specimens for spatial proteogenomic analysis.

Metronomic dosing of ovarian cancer cells with the ATR inhibitor AZD6738 leads to loss of CDC25A expression and resistance to ATRi treatment.

OBJECTIVE: ATR kinase inhibitors promote cell killing by inducing replication stress and through potentiation of genotoxic agents in gynecologic cancer cells. To explore mechanisms of acquired resistance to ATRi in ovarian cancer, we characterized ATRi-resistant ovarian cancer cells generated by metronomic dosing with the clinical ATR inhibitor AZD6738. METHODS: ATRi-resistant ovarian cancer cells (OVCAR3 and OV90) were generated by dosing with AZD6738 and assessed for sensitivity to Chk1i (LY2603618), PARPi (Olaparib) and combination with cisplatin or a CDK4/6 inhibitor (Palbociclib). Models were characterized by diverse methods including silencing CDC25A in OV90 cells and assessing impact on ATRi response. Serum proteomic analysis of ATRi-resistant OV90 xenografts was performed to identify circulating biomarker candidates of ATRi-resistance. RESULTS: AZD6738-resistant cell lines are refractory to LY2603618, but not to Olaparib or combinations with cisplatin. Cell cycle analyses showed ATRi-resistant cells exhibit G1/S arrest following AZD6738 treatment. Accordingly, combination with Palbociclib confers resistance to AZD6738. AZD6738-resistant cells exhibit altered abundances of G1/S phase regulatory proteins, including loss of CDC25A in AZD6738-resistant OV90 cells. Silencing of CDC25A in OV90 cells confers resistance to AZD6738. Serum proteomics from AZD6738-resistant OV90 xenografts identified Vitamin D-Binding Protein (GC), Apolipoprotein E (APOE) and A1 (APOA1) as significantly elevated in AZD6738-resistant backgrounds. CONCLUSIONS: We show that metronomic dosing of ovarian cancer cells with AZD6738 results in resistance to ATR/ Chk1 inhibitors, that loss of CDC25A expression represents a mechanism of resistance to ATRi treatment in ovarian cancer cells and identify several circulating biomarker candidates of CDC25A low, AZD6738-resistant ovarian cancer cells.

Improving Risk Assessment for Metastatic Disease in Endometrioid Endometrial Cancer Patients Using Molecular and Clinical Features: An NRG Oncology/Gynecologic Oncology Group Study.

Objectives: A risk assessment model for metastasis in endometrioid endometrial cancer (EEC) was developed using molecular and clinical features, and prognostic association was examined. Methods: Patients had stage I, IIIC, or IV EEC with tumor-derived RNA-sequencing or microarray-based data. Metastasis-associated transcripts and platform-centric diagnostic algorithms were selected and evaluated using regression modeling and receiver operating characteristic curves. Results: Seven metastasis-associated transcripts were selected from analysis in the training cohorts using 10-fold cross validation and incorporated into an MS7 classifier using platform-specific coefficients. The predictive accuracy of the MS7 classifier in Training-1 was superior to that of other clinical and molecular features, with an area under the curve (95% confidence interval) of 0.89 (0.80-0.98) for MS7 compared with 0.69 (0.59-0.80) and 0.71 (0.58-0.83) for the top evaluated clinical and molecular features, respectively. The performance of MS7 was independently validated in 245 patients using RNA sequencing and in 81 patients using microarray-based data. MS7 + MI (myometrial invasion) was preferrable to individual features and exhibited 100% sensitivity and negative predictive value. The MS7 classifier was associated with lower progression-free and overall survival (p ≤ 0.003). Conclusion: A risk assessment classifier for metastasis and prognosis in EEC patients with primary tumor derived MS7 + MI is available for further development and optimization as a companion clinical support tool.

Obesity and altered angiogenic-related gene expression in endometrial cancer.

OBJECTIVES: Evaluate association between obesity and angiogenic-related gene expression in endometrial cancer (EC). Evaluate interaction between diet and metformin on angiogenic-related gene expression. METHODS: We evaluated the association between 168 human angiogenic-related genes and body mass index (BMI) in the TCGA Uterine Corpus Endometrial Carcinoma cohort (endometrioid endometrial cancer (EEC) cohort n = 290, and copy number high cohort n = 55), an independent validation cohort from Gynecologic Cancer Center of Excellence (GYN-COE) (n = 62) and corresponding 185 homologous mouse genes in an LKB1fl/flp53fl/fl mouse model of EC (n = 20). Mice received 60% of calories from fat in a high-fat diet (HFD), mimicking diet-induced obesity, versus 10% of calories from fat in a low-fat diet (LFD). After tumor growth, HFD (n = 5) and LFD (n = 5) mice were treated with metformin (200 mg/kg/day) or control. Whole transcriptome analysis of mouse tumors was performed using RNA-Seq. RESULTS: At a false-discovery rate of 10%, twenty-one angiogenic-related genes were differentially expressed with respect to BMI when adjusting for grade in the TCGA EEC cohort. Evaluation of these genes in the mouse model control group revealed association between increased Edil3 expression in HFD versus LFD mice (2.5-fold change (FC); unadjusted p = 0.03). An interaction was observed for expression of Edil3 between diet and metformin treatment (unadjusted p = 0.009). Association between BMI and increased expression of EDIL3 was validated in one of four EDIL3 probesets in the GYN-COE cohort (p = 0.0011, adjusted p = 0.0342). CONCLUSIONS: Obesity may promote tumor progression via differential modulation of angiogenic pathways in EEC. Our exploratory findings demonstrated that EDIL3 may be a candidate gene of interest.

Extensive three-dimensional intratumor proteomic heterogeneity revealed by multiregion sampling in high-grade serous ovarian tumor specimens.

Enriched tumor epithelium, tumor-associated stroma, and whole tissue were collected by laser microdissection from thin sections across spatially separated levels of ten high-grade serous ovarian carcinomas (HGSOCs) and analyzed by mass spectrometry, reverse phase protein arrays, and RNA sequencing. Unsupervised analyses of protein abundance data revealed independent clustering of an enriched stroma and enriched tumor epithelium, with whole tumor tissue clustering driven by overall tumor "purity." Comparing these data to previously defined prognostic HGSOC molecular subtypes revealed protein and transcript expression from tumor epithelium correlated with the differentiated subtype, whereas stromal proteins (and transcripts) correlated with the mesenchymal subtype. Protein and transcript abundance in the tumor epithelium and stroma exhibited decreased correlation in samples collected just hundreds of microns apart. These data reveal substantial tumor microenvironment protein heterogeneity that directly bears on prognostic signatures, biomarker discovery, and cancer pathophysiology and underscore the need to enrich cellular subpopulations for expression profiling.

Proteogenomic landscape of uterine leiomyomas from hereditary leiomyomatosis and renal cell cancer patients.

Pathogenic mutations in fumarate hydratase (FH) drive hereditary leiomyomatosis and renal cell cancer (HLRCC) and increase the risk of developing uterine leiomyomas (ULMs). An integrated proteogenomic analysis of ULMs from HLRCC (n = 16; FH-mutation confirmed) and non-syndromic (NS) patients (n = 12) identified a significantly higher protein:transcript correlation in HLRCC (R = 0.35) vs. NS ULMs (R = 0.242, MWU p = 0.0015). Co-altered proteins and transcripts (228) included antioxidant response element (ARE) target genes, such as thioredoxin reductase 1 (TXNRD1), and correlated with activation of NRF2-mediated oxidative stress response signaling in HLRCC ULMs. We confirm 185 transcripts previously described as altered between HLRCC and NS ULMs, 51 co-altered at the protein level and several elevated in HLRCC ULMs are involved in regulating cellular metabolism and glycolysis signaling. Furthermore, 367 S-(2-succino)cysteine peptides were identified in HLRCC ULMs, of which sixty were significantly elevated in HLRCC vs. NS ULMs (LogFC = 1.86, MWU p < 0.0001). These results confirm and define novel proteogenomic alterations in uterine leiomyoma tissues collected from HLRCC patients and underscore conserved molecular alterations correlating with inactivation of the FH tumor suppressor gene.

Race-specific molecular alterations correlate with differential outcomes for black and white endometrioid endometrial cancer patients.

BACKGROUND: The objective of this study was to identify molecular alterations associated with disease outcomes for white and black patients with endometrioid endometrial cancer (EEC). METHODS: EEC samples from black (n = 17) and white patients (n = 13) were analyzed by proteomics (liquid chromatography-tandem mass spectrometry) and transcriptomics (RNA-seq). Coordinate alterations were validated with RNA-seq data from black (n = 49) and white patients (n = 216). Concordantly altered candidates were further tested for associations with race-specific progression-free survival (PFS) in black (n = 64) or white patients (n = 267) via univariate and multivariate Cox regression modeling and log-rank testing. RESULTS: Discovery analyses revealed significantly altered candidate proteins and transcripts between black and white patients, suggesting modulation of tumor cell viability in black patients and cell death signaling in black and white patients. Eighty-nine candidates were validated as altered between these patient cohorts, and a subset significantly correlated with differential PFS. White-specific PFS candidates included serpin family A member 4 (SERPINA4; hazard ratio [HR], 0.89; Wald P value = .02), integrin subunit α3 (ITGA3; HR, 0.76; P = .03), and Bet1 Golgi vesicular membrane trafficking protein like (BET1L; HR, 0.48; P = .04). Black-specific PFS candidates included family with sequence similarity 228 member B (FAM228B; HR, 0.13; P = .001) and HEAT repeat containing 6 (HEATR6; HR, 4.94; P = .047). Several candidates were also associated with overall survival (SERPINA4 and ITGA3) as well as PFS independent of disease stage, grade and myometrial invasion (SERPINA4, BET1L and FAM228B). CONCLUSIONS: This study has identified and validated molecular alterations in tumors from black and white EEC patients, including candidates significantly associated with altered disease outcomes within these patient cohorts. Cancer 2017;123:4004-12. © 2017 American Cancer Society.

Establishment and characterization of a platinum- and paclitaxel-resistant high grade serous ovarian carcinoma cell line.

High grade serous ovarian cancer (HGSOC) patients have a high recurrence rate after surgery and adjuvant chemotherapy due to inherent or acquired drug resistance. Cell lines derived from HGSOC tumors that are resistant to chemotherapeutic agents represent useful pre-clinical models for drug discovery. Here, we describe establishment of a human ovarian carcinoma cell line, which we term WHIRC01, from a patient-derived mouse xenograft established from a chemorefractory HGSOC patient who did not respond to carboplatin and paclitaxel therapy. This newly derived cell line is platinum- and paclitaxel-resistant with cisplatin, carboplatin, and paclitaxel half-maximal lethal doses of 15, 130, and 20 µM, respectively. Molecular characterization of this cell line was performed using targeted DNA exome sequencing, transcriptomics (RNA-seq), and mass spectrometry-based proteomic analyses. Results from exomic sequencing revealed mutations in TP53 consistent with HGSOC. Transcriptomic and proteomic analyses of WHIRC01 showed high level of alpha-enolase and vimentin, which are associated with cell migration and epithelial-mesenchymal transition. WHIRC01 represents a chemorefractory human HGSOC cell line model with a comprehensive molecular profile to aid future investigations of drug resistance mechanisms and screening of chemotherapeutic agents.

NUAK1 (ARK5) Is Associated with Poor Prognosis in Ovarian Cancer.

BACKGROUND AND OBJECTIVE: Nua kinase 1 (NUAK1) was identified in multigene signatures of survival and suboptimal debulking in high-grade serous ovarian cancer (HGSOC). This study investigates the individual clinical and biologic contributions of NUAK1 in HGSOC patients and cell lines. METHODS: Public transcript expression, clinical, and outcome data were used to interrogate the relationship between NUAK1 and clinicopathologic factors and patient outcomes including progression-free survival (PFS) and molecular subtypes using logistic and Cox modeling. Analysis of NUAK1 transcript expression was performed in primary tumors from 34 HGSOC patients with < or ≥2 years PFS. The impact of silencing NUAK1 by RNA interference (RNAi) on the migratory potential and chemosensitivity of SOC cells was assessed in vitro. RESULTS: Elevated NUAK1 transcript expression was associated with worse PFS (hazard ratio = 1.134), advanced stage (odds ratio, OR = 1.7), any residual disease (OR = 1.58), and mesenchymal disease subtype (OR = 7.79 ± 5.89). Elevated NUAK1 transcript expression was observed in HGSOC patients with < vs. ≥2 years PFS (p < 0.045). RNAi-mediated silencing of NUAK1 expression attenuated migration of OV90 and E3 HGSOC cells in vitro, but did not modulate sensitivity to cisplatin or paclitaxel. CONCLUSION: Elevated NUAK1 was associated with poor survival as well as advanced stage, residual disease after cytoreductive surgery and mesenchymal molecular subtype. NUAK1 impacted migration, but not chemosensitivity, in vitro. Additional studies are needed to further develop the concept of NUAK1 as a clinically deployable biomarker and therapeutic target in HGSOC.

Elevated AKAP12 in paclitaxel-resistant serous ovarian cancer cells is prognostic and predictive of poor survival in patients.

A majority of high-grade (HG) serous ovarian cancer (SOC) patients develop resistant disease despite high initial response rates to platinum/paclitaxel-based chemotherapy. We identified shed/secreted proteins in preclinical models of paclitaxel-resistant human HGSOC models and correlated these candidate proteins with patient outcomes using public data from HGSOC patients. Proteomic analyses of a HGSOC cell line secretome was compared to those from a syngeneic paclitaxel-resistant variant and from a line established from an intrinsically chemorefractory HGSOC patient. Associations between the identified candidate proteins and patient outcome were assessed in a discovery cohort of 545 patients and two validation cohorts totaling 795 independent SOC patients. Among the 81 differentially abundant proteins identified (q < 0.05) from paclitaxel-sensitive vs -resistant HGSOC cell secretomes, AKAP12 was verified to be elevated in all models of paclitaxel-resistant HGSOC. Furthermore, elevated AKAP12 transcript expression was associated with worse progression-free and overall survival. Associations with outcome were observed in three independent cohorts and remained significant after adjusted multivariate modeling. We further provide evidence to support that differential gene methylation status is associated with elevated expression of AKAP12 in taxol-resistant ovarian cancer cells and ovarian cancer patient subsets. Elevated expression and shedding/secretion of AKAP12 is characteristic of paclitaxel-resistant HGSOC cells, and elevated AKAP12 transcript expression is a poor prognostic and predictive marker for progression-free and overall survival in SOC patients.

MicroRNAs in endometrial cancers from black and white patients.

OBJECTIVE: Previous studies have identified differences in gene mutations among endometrial cancers from whites and blacks suggesting that differences in tumor biology may explain racial disparities in patient outcome. Micro RNAs (miRNAs) have emerged as regulators of transcript expression and their aberrant expression has been discovered in many diseases, including endometrial cancer. We performed quantitative polymerase chain reaction-based analysis in a set of endometrial cancers to identify whether there are racial differences in miRNA expression. STUDY DESIGN: Tumor cells from 50 stage-I endometrioid endometrial cancer specimens from 41 white and 9 black patients were prepared by laser microdissection and miRNA extracts were analyzed using TaqMan (Life Technologies, Carlsbad, CA) low-density arrays. Statistically significant, differentially expressed miRNAs between blacks and whites were identified using multidimensional scaling, Wilcoxon testing, and analysis of variance. RESULTS: There were no global differences in miRNA expression between endometrial cancers from 41 white and 9 black patients. To minimize potential bias introduced by unbalanced sample size, we performed a subset analysis with stage- and histology-matched specimens from 9 whites and 9 blacks that identified 18 differentially abundant miRNAs (>2-fold at P < .005). Quantitative polymerase chain reaction validated miRNA-337-3p in an independent set of endometrial cancer specimens from 23 white and 24 black women. There were no racial differences in hsa-miR-337-3p expression in normal endometrium. CONCLUSION: These data indicate that hsa-mir-337-3p is more frequently down-regulated in endometrial cancers from whites compared to blacks. Future studies are focused on determining the phenotypic impact of miR-337-3p and whether its differential expression is associated with clinical outcome.

Gene expression analysis of early stage endometrial cancers reveals unique transcripts associated with grade and histology but not depth of invasion.

Endometrial cancer is the most common gynecologic malignancy in the United States but it remains poorly understood at the molecular level. This investigation was conducted to specifically assess whether gene expression changes underlie the clinical and pathologic factors traditionally used for determining treatment regimens in women with stage I endometrial cancer. These include the effect of tumor grade, depth of myometrial invasion and histotype. We utilized oligonucleotide microarrays to assess the transcript expression profile in epithelial glandular cells laser microdissected from 79 endometrioid and 12 serous stage I endometrial cancers with a heterogeneous distribution of grade and depth of myometrial invasion, along with 12 normal post-menopausal endometrial samples. Unsupervised multidimensional scaling analyses revealed that serous and endometrioid stage I cancers have similar transcript expression patterns when compared to normal controls where 900 transcripts were identified to be differentially expressed by at least fourfold (univariate t-test, p < 0.001) between the cancers and normal endometrium. This analysis also identified transcript expression differences between serous and endometrioid cancers and tumor grade, but no apparent differences were identified as a function of depth of myometrial invasion. Four genes were validated by quantitative PCR on an independent set of cancer and normal endometrium samples. These findings indicate that unique gene expression profiles are associated with histologic type and grade, but not myometrial invasion among early stage endometrial cancers. These data provide a comprehensive perspective on the molecular alterations associated with stage I endometrial cancer, particularly those subtypes that have the worst prognosis.

Transcript expression in endometrial cancers from Black and White patients.

OBJECTIVE: Previous studies suggest that differences in molecular features of endometrial cancers between racial groups may contribute to the poorer survival in Blacks. The objective of this investigation was to determine whether gene expression among endometrial cancers is different between Blacks and Whites. METHODS: Fresh frozen tumors from 25 Black patients were matched by stage, grade, and histology to endometrial cancer specimens from 25 White patients. Each case was macrodissected to produce specimens possessing a minimum of 75% cancer cellularity. A subset of 10 matched pairs was also prepared using laser microdissection (LMD) to produce specimens possessing a minimum of 95% cancer cells. Total RNA isolated from each sample was analyzed using the Affymetrix Human Genome U133 Plus 2.0 arrays. Data were analyzed using principal component analysis and binary class comparison analyses. RESULTS: Unsupervised analysis of the 50 endometrial cancers failed to identify global gene expression profiles unique to Black or White patients. In a subset analysis of 10 matched pairs from Blacks and Whites prepared using LMD and macrodissection, unsupervised analysis did not reveal a unique gene expression profile associated with race in either set, but associations were identified that relate to sample preparation technique, histology and stage. CONCLUSIONS: Our microarray data revealed no global gene expression differences and identified few individual gene differences between endometrial cancers from Blacks and Whites. More comprehensive methods of transcriptome analysis could uncover RNAs that may underpin the disparity of outcome or prevalence of endometrial cancers in Blacks and Whites.

Analysis of PSPHL as a Candidate Gene Influencing the Racial Disparity in Endometrial Cancer.

Endometrial cancer is the most commonly diagnosed gynecologic malignancy in the United States. A well recognized disparity by race in both incidence and survival outcome exists for this cancer. Specifically Caucasians are about two times more likely to develop endometrial cancer than are African-Americans. However, African-American women are more likely to die from this disease than are Caucasians. The basis for this disparity remains unknown. Previous studies have identified differences in the types and frequencies of gene mutations among endometrial cancers from Caucasians and African-Americans suggesting that the tumors from these two groups might have differing underlying genetic defects. We performed a gene expression microarray study in an effort to identify differentially expressed transcripts between African-American and Caucasian women's endometrial cancers. Our gene expression screen identified a list of potential biomarkers that are differentially expressed between these two groups of cancers. Of these we identified a poorly characterized transcript with a region of homology to phospho serine phosphatase (PSPH) and designated phospho serine phosphatase like (PSPHL) as the most differentially over-expressed gene in cancers from African-Americans. We further clarified the nature of expressed transcripts. Northern blot analysis confirmed the message was limited to a transcript of under 1 kB. Sequence analysis of transcripts confirmed two alternate open reading frame (ORF) isoforms due to alternative splicing events. Splice specific primer sets confirmed both isoforms were differentially expressed in tissues from Caucasians and African-Americans. We further examined the expression in other tissues from women to include normal endometrium, normal and malignant ovary. In all cases PSPHL expression was more often present in tissues from African-Americans than Caucasians. Our data confirm the African-American based expression of the PSPHL transcript in endometrial cancer and also identify its expression in other tissues from African-Americans including ovary and ovarian cancer. PSPHL represents a candidate gene that might influence the observed racial disparity in endometrial and other cancers.

Proteomic analysis of stage I endometrial cancer tissue: identification of proteins associated with oxidative processes and inflammation.

OBJECTIVE: The present study aimed to identify differentially expressed proteins employing a high resolution mass spectrometry (MS)-based proteomic analysis of endometrial cancer cells harvested using laser microdissection. METHODS: A differential MS-based proteomic analysis was conducted from discrete epithelial cell populations gathered by laser microdissection from 91 pathologically reviewed stage I endometrial cancer tissue samples (79 endometrioid and 12 serous) and 10 samples of normal endometrium from postmenopausal women. Hierarchical cluster analysis of protein abundance levels derived from a spectral count analysis revealed a number of proteins whose expression levels were common as well as unique to both histologic types. An independent set of endometrial cancer specimens from 394 patients were used to externally validate the differential expression of select proteins. RESULTS: 209 differentially expressed proteins were identified in a comparison of stage I endometrial cancers and normal post-menopausal endometrium controls (Q<0.005). A number of differentially abundant proteins in stage I endometrial cancer were identified and independently validated by western blot and tissue microarray analyses. Multiple proteins identified with elevated abundance in stage I endometrial cancer are functionally associated with inflammation (annexins) and oxidative processes (peroxiredoxins). PRDX1 and ANXA2 were both confirmed as being overexpressed in stage I cancer compared to normal endometrium by independent TMA (Q=0.008 and Q=0.00002 respectively). CONCLUSIONS: These data provide the basis for further investigation of previously unrecognized novel pathways involved in early stage endometrial carcinogenesis and provide possible targets for prevention strategies that are inclusive of both endometrioid and serous histologic subtypes.

Global expression analysis of cancer/testis genes in uterine cancers reveals a high incidence of BORIS expression.

PURPOSE: Cancer/testis (CT) genes predominantly expressed in the testis (germ cells) and generally not in other normal tissues are aberrantly expressed in human cancers. This highly restricted expression provides a unique opportunity to use these CT genes for diagnostics, immunotherapeutic, or other targeted therapies. The purpose of this study was to identify those CT genes with the greatest incidence of expression in uterine cancers. EXPERIMENTAL DESIGN: We queried the expression of known and putative CT gene transcripts (representing 79 gene loci) using whole genome gene expression arrays. Specifically, the global gene expressions of uterine cancers (n = 122) and normal uteri (n = 10) were determined using expression data from the Affymetrix HG-U133A and HG-U133B chips. Additionally, we also examined the brother of the regulator of imprinted sites (BORIS) transcript by reverse transcription-PCR and quantitative PCR because its transcript was not represented on the array. RESULTS: Global microarray analysis detected many CT genes expressed in various uterine cancers; however, no individual CT gene was expressed in more than 25% of all cancers. The expression of the two most commonly expressed CT genes on the arrays, MAGEA9 (24 of 122 cancers and 0 of 10 normal tissues) and Down syndrome critical region 8 (DSCR8)/MMA1 (16 if 122 cancers and 0 of 10 normal tissues), was confirmed by reverse transcription-PCR methods, validating the array screening approach. In contrast to the relatively low incidence of expression of the other CT genes, BORIS expression was detected in 73 of 95 (77%) endometrial cancers and 24 of 31 (77%) uterine mixed mesodermal tumors. CONCLUSIONS: These data provide the first extensive survey of multiple CT genes in uterine cancers. Importantly, we detected a high frequency of BORIS expression in uterine cancers, suggesting its potential as an immunologic or diagnostic target for these cancers. Given the high incidence of BORIS expression and its possible regulatory role, an examination of BORIS function in the etiology of these cancers is warranted.