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ImportanceDespite similar histologic appearance among high-grade serous ovarian cancers (HGSOCs), clinical observations suggest vast differences in gross appearance. There is currently no systematic framework by which to classify HGSOCs according to their gross morphologic characteristics.ObjectiveTo develop and characterize a gross morphologic classification system for HGSOC.Design, Setting, and ParticipantsThis cohort study included patients with suspected advanced-stage ovarian cancer who presented between April 1, 2013, and August 5, 2016, to the University of Texas MD Anderson Cancer Center, a large referral center. Patients underwent laparoscopic assessment of disease burden before treatment and received a histopathologic diagnosis of HGSOC. Researchers assigning morphologic subtype and performing molecular analyses were blinded to clinical outcomes. Data analysis was performed between April 2020 and November 2021.ExposuresGross tumor morphologic characteristics.Main Outcomes and MeasuresClinical outcomes and multiomic profiles of representative tumor samples of type I or type II morphologic subtypes were compared.ResultsOf 112 women (mean [SD] age 62.7 [9.7] years) included in the study, most patients (84% [94]) exhibited a predominant morphologic subtype and many (63% [71]) had a uniform morphologic subtype at all involved sites. Compared with those with uniform type I morphologic subtype, patients with uniform type II morphologic subtype were more likely to have a favorable Fagotti score (83% [19 of 23] vs 46% [22 of 48]; P = .004) and thus to be triaged to primary tumor reductive surgery. Similarly, patients with uniform type II morphologic subtype also had significantly higher mean (SD) estimated blood loss (639 [559; 95% CI, 391-887] mL vs 415 [527; 95% CI, 253-577] mL; P = .006) and longer mean (SD) operative time (408 [130; 95% CI, 350-466] minutes vs 333 [113; 95% CI, 298-367] minutes; P = .03) during tumor reductive surgery. Type I tumors had enrichment of epithelial-mesenchymal transition (false discovery rate [FDR] q-value, 3.10 × 10−24), hypoxia (FDR q-value, 1.52 × 10−5), and angiogenesis pathways (FDR q-value, 2.11 × 10−2), whereas type II tumors had enrichment of pathways related to MYC signaling (FDR q-value, 2.04 × 10−9) and cell cycle progression (FDR q-value, 1.10 × 10−5) by integrated proteomic and transcriptomic analysis. Abundances of metabolites and lipids also differed between the 2 morphologic subtypes.Conclusions and RelevanceThis study identified 2 novel, gross morphologic subtypes of HGSOC, each with unique clinical features and molecular signatures. The findings may have implications for triaging patients to surgery or chemotherapy, identifying outcomes, and developing tailored therapeutic strategies.

Background Low-grade serous ovarian cancer (LGSOC) is a rare disease that occurs more frequently in younger women than those with high-grade disease. The current treatment is suboptimal and a better understanding of the molecular pathogenesis of this disease is required. In this study, we compared the proteogenomic analyses of LGSOCs from short- and long-term survivors (defined as 60 months, respectively). Our goal was to identify novel mutations, proteins, and mRNA transcripts that are dysregulated in LGSOC, particularly in short-term survivors. Methods Initially, targeted sequencing of 409 cancer-related genes was performed on 22 LGSOC and 6 serous borderline ovarian tumor samples. Subsequently, whole-genome sequencing analysis was performed on 14 LGSOC samples (7 long-term survivors and 7 short-term survivors) with matched normal tissue samples. RNA sequencing (RNA-seq), quantitative proteomics, and phosphoproteomic analyses were also performed. Results We identified single-nucleotide variants (SNVs) (range: 5688–14,833 per sample), insertion and deletion variants (indels) (range: 880–1065), and regions with copy number variants (CNVs) (range: 62–335) among the 14 LGSOC samples. Among all SNVs and indels, 2637 mutation sites were found in the exonic regions. The allele frequencies of the detected variants were low (median12%). The identified recurrent nonsynonymous missense mutations included KRAS, NRAS, EIF1AX, UBR5, and DNM3 mutations. Mutations in DNM3 and UBR5 have not previously been reported in LGSOC. For the two samples, somatic DNM3 nonsynonymous missense mutations in the exonic region were validated using Sanger sequencing. The third sample contained two missense mutations in the intronic region of DNM3, leading to a frameshift mutation detected in RNA transcripts in the RNA-seq data. Among the 14 LGSOC samples, 7754 proteins and 9733 phosphosites were detected by global proteomic analysis. Some of these proteins and signaling pathways, such as BST1, TBXAS1, MPEG1, HBA1, and phosphorylated ASAP1, are potential therapeutic targets. Conclusions This is the first study to use whole-genome sequencing to detect somatic mutations in LGSOCs with matched normal tissues. We detected and validated novel mutations in DNM3, which were present in 3 of the 14 samples analyzed. Additionally, we identified novel indels, regions with CNVs, dysregulated mRNA, dysregulated proteins, and phosphosites that are more prevalent in short-term survivors. This integrated proteogenomic analysis can guide research into the pathogenesis and treatment of LGSOC.

BACKGROUND: Homologous recombination deficiency (HRD) score is related to chemotherapy response in some cancers, but its role in endometrial cancer in not known. We determined frequency and clinical significance of alterations in the HR pathway in endometrial cancer. METHODS: 253 endometrioid endometrial adenocarcinoma (EEA) samples from two independent cohorts (discovery and replication) were tested for HRD score using the Myriad HRD assay, microsatellite instability (MSI) and tumor mutation burden (TMB) using a next generation sequencing assay. HRD scores were also generated on endometrial cancer cell lines and in vivo response to olaparib was assessed. RESULTS: ROC curves were employed to determine optimal cutoffs of HRD in relation to survival impact in endometrial cancer and a cutoff of HRD ≥ 4 was suggested for DFS using discovery cohort. Patients from two independent cohorts with HRD score ≥ 4 trended toward worse survival as compared to those with HRD score

The tumor microenvironment (TME) represents a complex ecosystem comprised of dozens of distinct cell types, including tumor, stroma, and immune cell populations. To characterize proteome-level variation and tumor heterogeneity at scale, high-throughput methods are needed to selectively isolate discrete cellular populations in solid tumor malignancies. This protocol describes a high-throughput workflow, enabled by artificial intelligence (AI), that segments images of hematoxylin and eosin (HE)-stained, thin tissue sections into pathology-confirmed regions of interest for selective harvest of histology-resolved cell populations using laser microdissection (LMD). This strategy includes a novel algorithm enabling the transfer of regions denoting cell populations of interest, annotated using digital image software, directly to laser microscopes, thus enabling more facile collections. Successful implementation of this workflow was performed, demonstrating the utility of this harmonized method to selectively harvest tumor cell populations from the TME for quantitative, multiplexed proteomic analysis by high-resolution mass spectrometry. This strategy fully integrates with routine histopathology review, leveraging digital image analysis to support enrichment of cellular populations of interest and is fully generalizable, enabling harmonized harvests of cell populations from the TME for multiomic analyses.

We present a deep proteogenomic profiling study of 87 lung adenocarcinoma (LUAD) tumors from the United States, integrating whole-genome sequencing, transcriptome sequencing, proteomics and phosphoproteomics by mass spectrometry, and reverse-phase protein arrays. We identify three subtypes from somatic genome signature analysis, including a transition-high subtype enriched with never smokers, a transversion-high subtype enriched with current smokers, and a structurally altered subtype enriched with former smokers, TP53 alterations, and genome-wide structural alterations. We show that within-tumor correlations of RNA and protein expression associate with tumor purity and immune cell profiles. We detect and independently validate expression signatures of RNA and protein that predict patient survival. Additionally, among co-measured genes, we found that protein expression is more often associated with patient survival than RNA. Finally, integrative analysis characterizes three expression subtypes with divergent mutations, proteomic regulatory networks, and therapeutic vulnerabilities. This proteogenomic characterization provides a foundation for molecularly informed medicine in LUAD.

Preoperative use of metformin in obese women with endometrioid endometrial cancer (EEC) reduces tumor proliferation and inhibits the mammalian target of rapamycin pathway, though is only effective in select cases. This study sought to identify a predictive and/or pharmacodynamic proteomic signature of metformin response to tailor its pharmacologic use. Matched pre‐ and post‐metformin‐treated tumor tissues from a recently completed preoperative window trial of metformin in EEC patients (ClinicalTrials.gov: NCT01911247) were analyzed by mass spectrometry (MS)‐based proteomic and immunohistochemical analyses. Jupiter microtubule‐associated homolog 1 (JPT1) was significantly elevated in metformin responders (n = 13) vs nonresponders (n = 7), and found to decrease in abundance in metformin responders following treatment; observations that were verified by immunohistochemical staining for JPT1. Metformin response and loss of JPT1 were assessed in RL95‐2 and ACI‐181 endometrial cancer (EC) cell lines. We further identified that silencing of JPT1 abundance does not alter cellular response to metformin or basal cell proliferation, but that JPT1 abundance does decrease in response to metformin treatment in RL95‐2 and ACI‐181 EC cell lines. These data suggest that JPT1 represents a predictive and pharmacodynamic biomarker of metformin response that, if validated in larger patient populations, may enable preoperative EEC patient stratification to metformin treatment and the ability to monitor patient response., We report JPT1 (Jupiter microtubule‐associated homolog 1) as a predictive and pharmacodynamic biomarker candidate of metformin response in endometrial cancers.

Develop conditional survival and risk-assessment estimates for uterine serous carcinoma (USC) overall and stratified by stage as tools for annual survivorship counseling and care planning.Patients in the National Cancer Data Base diagnosed between 2004 and 2014 with stage I-IV USC were eligible. Individuals missing stage or survival data or with multiple malignancies were excluded. Five-year conditional survival was estimated using the stage-stratified Kaplan-Meier method annually during follow-up. A standardized mortality ratio (SMR) estimated the proportion of observed to expected deaths in the U.S. adjusted for year, age, and race. The relationships between prognostic factors and survival were studied using multivariate Cox modeling at diagnosis and conditioned on surviving 5-years.There were 14,575 participants, including 43% with stage I, 8% with stage II, 29% with stage III, and 20% with stage IV USC. Five-year survival at diagnosis vs. after surviving 5-years was 52% vs. 75% overall, 77% vs. 81% for stage I, 57% vs. 72% for stage II, 40% vs. 66% for stage III, and 17% vs. 60% for stage IV USC, respectively (P0.0001). Incremental improvements in 5-year conditional survival and reductions in SMR tracked with annual follow-up and higher stage. The adjusted risk of death at diagnosis vs. after surviving 5-years was 1.15 vs. 1.40 per 5-year increase of age, 1.26 vs. 1.68 for Medicaid insurance, 3.92 vs. 2.48 for stage III disease, and 6.65 vs. 2.79 for stage IV disease, respectively (P0.0001).In USC, the evolution of conditional survival permits annual reassessments of prognosis to tailor survivorship counseling and care planning.

All human diseases involve proteins, yet our current tools to characterize and quantify them are limited. To better elucidate proteins across space, time, and molecular composition, we provide a >10 years of projection for technologies to meet the challenges that protein biology presents. With a broad perspective, we discuss grand opportunities to transition the science of proteomics into a more propulsive enterprise. Extrapolating recent trends, we describe a next generation of approaches to define, quantify, and visualize the multiple dimensions of the proteome, thereby transforming our understanding and interactions with human disease in the coming decade.

Endosomal trafficking of cell surface receptors is essential to their function. Integrins are transmembrane receptors that integrate adhesion to the extracellular matrix with engagement of the cytoskeleton. Ligated integrins mediate diverse signals that regulate matrix assembly, cell survival, cell morphology, and cell motility. Endosomal trafficking of integrins modulates these signals and contributes to cell motility and is required for cancer cell invasion. The phosphoprotein PEA-15 modulates integrin activation and ERK MAP Kinase signaling. To elucidate novel PEA-15 functions we utilized an unbiased proteomics approach. We identified several binding partners for PEA-15 in the endosome including clathrin and AP-2 as well as integrin β1 and other focal adhesion complex proteins. We confirmed these interactions using proximity ligation analysis, immunofluorescence imaging, pull-down and co-immunoprecipitation. We further found that PEA-15 is enriched in endosomes and was required for efficient endosomal internalization of α5β1 integrin and cellular migration. Importantly, PEA-15 promotion of migration was dependent on PEA-15 phosphorylation at serines 104 and 116. These data support a novel endosomal role for PEA-15 in control of endosomal trafficking of integrins through an association with the β1 integrin and clathrin complexes, and thereby regulation of cell motility.

Tubo-ovarian high-grade serous carcinomas (HGSC) are highly proliferative neoplasms that generally respond well to platinum/taxane chemotherapy. We recently identified minichromosome maintenance complex component 3 (MCM3), which is involved in the initiation of DNA replication and proliferation, as a favorable prognostic marker in HGSC. Our objective was to further validate whether MCM3 mRNA expression and possibly MCM3 protein levels are associated with survival in patients with HGSC. MCM3 mRNA expression was measured using NanoString expression profiling on formalin-fixed and paraffin-embedded tissue (N = 2355 HGSC) and MCM3 protein expression was assessed by immunohistochemistry (N = 522 HGSC) and compared with Ki-67. Kaplan–Meier curves and the Cox proportional hazards model were used to estimate associations with survival. Among chemotherapy-naïve HGSC, higher MCM3 mRNA expression (one standard deviation increase in the score) was associated with longer overall survival (HR = 0.87, 95% CI 0.81–0.92, p < 0.0001, N = 1840) in multivariable analysis. MCM3 mRNA expression was highest in the HGSC C5.PRO molecular subtype, although no interaction was observed between MCM3, survival and molecular subtypes. MCM3 and Ki-67 protein levels were significantly lower after exposure to neoadjuvant chemotherapy compared to chemotherapy-naïve tumors: 37.0% versus 46.4% and 22.9% versus 34.2%, respectively. Among chemotherapy-naïve HGSC, high MCM3 protein levels were also associated with significantly longer disease-specific survival (HR = 0.52, 95% CI 0.36–0.74, p = 0.0003, N = 392) compared to cases with low MCM3 protein levels in multivariable analysis. MCM3 immunohistochemistry is a promising surrogate marker of proliferation in HGSC.

Activation of Pannexin 1 (PANX1) ion channels causes release of intercellular signaling molecules in a variety of (patho)physiological contexts. PANX1 can be activated by G protein-coupled receptors (GPCRs), including α1-adrenergic receptors (α1-ARs), but how receptor engagement leads to channel opening remains unclear. Here, we show that GPCR-mediated PANX1 activation can occur via channel deacetylation. We find that α1-AR-mediated activation of PANX1 channels requires Gαq but is independent of phospholipase C or intracellular calcium. Instead, α1-AR-mediated PANX1 activation involves RhoA, mammalian diaphanous (mDia)-related formin, and a cytosolic lysine deacetylase activated by mDia – histone deacetylase 6. HDAC6 associates with PANX1 and activates PANX1 channels, even in excised membrane patches, suggesting direct deacetylation of PANX1. Substitution of basally-acetylated intracellular lysine residues identified on PANX1 by mass spectrometry either prevents HDAC6-mediated activation (K140/409Q) or renders the channels constitutively active (K140R). These data define a non-canonical RhoA-mDia-HDAC6 signaling pathway for GαqPCR activation of PANX1 channels and uncover lysine acetylation-deacetylation as an ion channel silencing-activation mechanism., Pannexin 1 (PANX1) is a membrane channel mediating release of signaling molecules to the extracellular space. PANX1 can be activated by GPCRs. Here, the authors elucidate a non-canonical channel activation pathway by α1-adrenergic receptor that involves HDAC6- mediated lysine deacetylation of PANX1.

Although 70–80% of newly diagnosed ovarian cancer patients respond to first-line therapy, almost all relapse and five-year survival remains below 50%. One strategy to increase five-year survival is prolonging time to relapse by improving first-line therapy response. However, no biomarker today can accurately predict individual response to therapy. In this study, we present analytical and prospective clinical validation of a new test that utilizes primary patient tissue in 3D cell culture to make patient-specific response predictions prior to initiation of treatment in the clinic. Test results were generated within seven days of tissue receipt from newly diagnosed ovarian cancer patients obtained at standard surgical debulking or laparoscopic biopsy. Patients were followed for clinical response to chemotherapy. In a study population of 44, the 32 test-predicted Responders had a clinical response rate of 100% across both adjuvant and neoadjuvant treated populations with an overall prediction accuracy of 89% (39 of 44, p

Background E39, an HLA‐A2‐restricted, immunogenic peptide derived from the folate‐binding protein (FBP), is overexpressed in multiple malignancies. We conducted a phase I/IIa trial of the E39 + GM‐CSF vaccine with booster inoculations of either E39 or E39′ (an attenuated version of E39) to prevent recurrences in disease‐free endometrial and ovarian cancer patients(pts). Here, we present the final 24‐month landmark analysis. Patients and methods HLA‐A2 + patients receiving E39 + GM‐CSF were included in the vaccine group (VG), and HLA‐A2‐ pts (or HLA‐A2 + patients refusing vaccine) were followed as the control group (CG). VG group received 6 monthly inoculations as the primary vaccine series (PVS) and were randomized to receive either E39 or E39′ booster inoculations. Demographic, safety, immunologic, and disease‐free survival (DFS) data were collected and evaluated. Results Fifty‐one patients were enrolled; 29 in the VG and 22 in the CG. Fourteen patients received

Background Although cell lines are an essential resource for studying cancer biology, many are of unknown ancestral origin, and their use may not be optimal for evaluating the biology of all patient populations. Methods An admixture analysis was performed using genome‐wide chip data from the Catalogue of Somatic Mutations in Cancer (COSMIC) Cell Lines Project to calculate genetic ancestry estimates for 1018 cancer cell lines. After stratifying the analyses by tissue and histology types, linear models were used to evaluate the influence of ancestry on gene expression and somatic mutation frequency. Results For the 701 cell lines with unreported ancestry, 215 were of East Asian origin, 30 were of African or African American origin, and 453 were of European origin. Notable imbalances were observed in ancestral representation across tissue type, with the majority of analyzed tissue types having few cell lines of African American ancestral origin, and with Hispanic and South Asian ancestry being almost entirely absent across all cell lines. In evaluating gene expression across these cell lines, expression levels of the genes neurobeachin line 1 (NBEAL1), solute carrier family 6 member 19 (SLC6A19), HEAT repeat containing 6 (HEATR6), and epithelial cell transforming 2 like (ECT2L) were associated with ancestry. Significant differences were also observed in the proportions of somatic mutation types across cell lines with varying ancestral proportions. Conclusions By estimating genetic ancestry for 1018 cancer cell lines, the authors have produced a resource that cancer researchers can use to ensure that their cell lines are ancestrally representative of the populations they intend to affect. Furthermore, the novel ancestry‐specific signal identified underscores the importance of ancestral awareness when studying cancer., Preclinical cancer cell line research is often conducted without an awareness of ancestral background, which results in incongruities between the genetic backgrounds of used cell lines and the patient populations they are intended to represent. By calculating genetic ancestry for 1018 common cancer cell lines and identifying ancestry‐specific expression and somatic mutation patterns, the importance of ancestral awareness is emphasized, and a resource is provided that can be used by cancer researchers to ensure that their cell lines are ancestrally representative of the populations they aim to impact.

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.

Cancer has no borders: Generation and analysis of molecular data across multiple centers worldwide is necessary to gain statistically significant clinical insights for the benefit of patients. Here we conceived and standardized a proteotype data generation and analysis workflow enabling distributed data generation and evaluated the quantitative data generated across laboratories of the international Cancer Moonshot consortium. Using harmonized mass spectrometry (MS) instrument platforms and standardized data acquisition procedures, we demonstrate robust, sensitive, and reproducible data generation across eleven international sites on seven consecutive days in a 24/7 operation mode. The data presented from the high-resolution MS1-based quantitative data-independent acquisition (HRMS1-DIA) workflow shows that coordinated proteotype data acquisition is feasible from clinical specimens using such standardized strategies. This work paves the way for the distributed multi-omic digitization of large clinical specimen cohorts across multiple sites as a prerequisite for turning molecular precision medicine into reality., Distributed multi-omic digitization of clinical specimen across multiple sites is a prerequisite for turning molecular precision medicine into reality. Here, the authors show that coordinated proteotype data acquisition is feasible using standardized MS data acquisition and analysis strategies.

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 FH tumor suppressor gene.

Cesarean section (CS) is recognized as being a shared environmental risk factor associated with chronic immune disease. A study of maternal gene expression changes between different delivery modes can add to our understanding of how CS contributes to disease patterns later in life. We evaluated the association of delivery mode with postpartum gene expression using a cross-sectional study of 324 mothers who delivered full-term (≥ 37 weeks) singletons. Of these, 181 mothers had a vaginal delivery and 143 had a CS delivery (60 with and 83 without labor). Antimicrobial peptides (AMP) were upregulated in vaginal delivery compared to CS with or without labor. Peptidase inhibitor 3 (PI3), a gene in the antimicrobial peptide pathway and known to be involved in antimicrobial and anti-inflammatory activities, showed a twofold increase in vaginal delivery compared to CS with or without labor (adjusted p-value 1.57 × 10–11 and 3.70 × 10–13, respectively). This study evaluates differences in gene expression by delivery mode and provides evidence of antimicrobial peptide upregulation in vaginal delivery compared to CS with or without labor. Further exploration is needed to determine if AMP upregulation provides protection against CS-associated diseases later in life.

Background There is a critical need to identify patient characteristics associated with long-term ovarian cancer survival. Methods Quality of life (QOL), measured by the Functional Assessment of Cancer Therapy-Ovarian-Trial Outcome Index (FACT-O-TOI), including physical, functional, and ovarian-specific subscales, was compared between long-term survivors (LTS) (8+ years) and short-term survivors (STS) ( Results QOL differed statistically significantly between STS (N = 1115) and LTS (N = 260) (P Conclusions Baseline and longitudinal QOL change scores distinguished LTS vs STS and are robust prognosticators for long-term survival. Results have trial design and supportive care implications, providing meaningful prognostic value in this understudied population.

Objectives To investigate the association between CT imaging traits and texture metrics with proteomic data in patients with high-grade serous ovarian cancer (HGSOC). Methods This retrospective, hypothesis-generating study included 20 patients with HGSOC prior to primary cytoreductive surgery. Two readers independently assessed the contrast-enhanced computed tomography (CT) images and extracted 33 imaging traits, with a third reader adjudicating in the event of a disagreement. In addition, all sites of suspected HGSOC were manually segmented texture features which were computed from each tumor site. Three texture features that represented intra- and inter-site tumor heterogeneity were used for analysis. An integrated analysis of transcriptomic and proteomic data identified proteins with conserved expression between primary tumor sites and metastasis. Correlations between protein abundance and various CT imaging traits and texture features were assessed using the Kendall tau rank correlation coefficient and the Mann-Whitney U test, whereas the area under the receiver operating characteristic curve (AUC) was reported as a metric of the strength and the direction of the association. P values Results Four proteins were associated with CT-based imaging traits, with the strongest correlation observed between the CRIP2 protein and disease in the mesentery (p p = 0.047, τ = 0.326). Conclusion This study provides the first insights into the potential associations between standard-of-care CT imaging traits and texture measures of intra- and inter-site heterogeneity, and the abundance of several proteins. Key Points • CT-based texture features of intra- and inter-site tumor heterogeneity correlate with the abundance of several proteins in patients with HGSOC. • CT imaging traits correlate with protein abundance in patients with HGSOC.

DNA-damaging chemotherapy and radiation therapy are integrated into the treatment paradigm of the majority of cancer patients. Recently, immunotherapy that targets the immunosuppressive interaction between programmed death 1 (PD-1) and its ligand PD-L1 has been approved for malignancies including non–small cell lung cancer, melanoma, and head and neck squamous cell carcinoma. ATR is a DNA damage–signaling kinase activated at damaged replication forks, and ATR kinase inhibitors potentiate the cytotoxicity of DNA-damaging chemotherapies. We show here that the ATR kinase inhibitor AZD6738 combines with conformal radiation therapy to attenuate radiation-induced CD8+ T cell exhaustion and potentiate CD8+ T cell activity in mouse models of Kras-mutant cancer. Mechanistically, AZD6738 blocks radiation-induced PD-L1 upregulation on tumor cells and dramatically decreases the number of tumor-infiltrating Tregs. Remarkably, AZD6738 combines with conformal radiation therapy to generate immunologic memory in complete responder mice. Our work raises the possibility that a single pharmacologic agent may enhance the cytotoxic effects of radiation while concurrently potentiating radiation-induced antitumor immune responses.

Racial differences in the molecular subtypes of endometrial cancer and associations with progression-free survival (PFS) were evaluated.Molecular, clinical and PFS data were acquired from the Cancer Genome Atlas (TCGA) including classification into the integrative, somatic copy number alteration and transcript-based subtypes. The prevalence and prognostic value of the aggressive molecular subtypes (copy number variant [CNV]-high, cluster 4 or mitotic) were evaluated in Black and White patients.There were 337 patients including 14% self-designated as Black, 27% with advanced stage, and 82% with endometrioid histology. The CNV-high subtype was more common in Black than White patients (61.9% vs. 23.5%, P=0.0005) and suggested worse PFS in Black patients (hazard ratio [HR]=3.4, P=0.189). The cluster 4 subtype was more prevalent in Black patients (56.8% vs. 20.9%, P0.0001) and associated with worse PFS in Black patients (HR=3.4, P=0.049). The mitotic subtype was more abundant in Black patients (64.1% vs. 33.7%, P=0.002), indicated worse PFS in Black patients (HR=4.1, P=0.044) including the endometrioid histology (HR=6.1, P=0.024) and exhibited race-associated enrichment in cell cycle signaling and pathways in cancer including PLK1 and BIRC7. All of these aggressive molecular subtypes also indicated worse PFS in White patients, with unique enrichments in mitotic signaling different from Black patients.The aggressive molecular subtypes from TCGA were more common in Black endometrial cancer patients and indicated worse PFS in both Black and White patients. The mitotic subtypes also indicated worse PFS in Black patients with endometrioid histology. Enrichment patterns in mitotic signaling may represent therapeutic opportunities.

Solid tumour malignancies comprise a highly variable admixture of tumour and non-tumour cellular populations, forming a complex cellular ecosystem and tumour microenvironment. This tumour heterogeneity is not incidental, and is known to correlate with poor patient prognosis for many cancer types. Indeed, non-malignant cell populations, such as vascular endothelial and immune cells, are known to play key roles supporting and, in some cases, driving aggressive tumour biology, and represent targets of emerging therapeutics, such as antiangiogenesis and immune checkpoint inhibitors. The biochemical interplay between these cellular populations and how they contribute to molecular tumour heterogeneity remains enigmatic, particularly from the perspective of the tumour proteome. This review focuses on recent advances in proteomic methods, namely imaging mass spectrometry, single-cell proteomic techniques, and preanalytical sample processing, that are uniquely positioned to enable detailed analysis of discrete cellular populations within tumours to improve our understanding of tumour proteomic heterogeneity. This review further emphasizes the opportunity afforded by the application of these techniques to the analysis of tumour heterogeneity in formalin-fixed paraffin-embedded archival tumour tissues, as these represent an invaluable resource for retrospective analyses that is now routinely accessible, owing to recent technological and methodological advances in tumour tissue proteomics. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Gestational choriocarcinoma is a malignant form of gestational trophoblastic disease that usually arises after a molar pregnancy, but may follow any antecedent pregnancy. Investigations in this rare cancer are limited. We evaluated the prognostic effects of age, race, and stage in choriocarcinomas diagnosed for 4 decades.Patients diagnosed as having gestational choriocarcinoma between 1973 and 2014 from the Surveillance, Epidemiology, and End Results program were eligible. Relationships with overall survival and cancer-specific survival were evaluated using log-rank testing and Cox modeling. Multivariate analyses included adjustments for age, race, and stage.There were 947 patients with choriocarcinoma including 403 non-Hispanic white (NHW) patients, 473 with distant stage, and 142 who died. Median age at diagnosis was 25 years for non-Hispanic black (NHB) patients and 35 years for Asian/Pacific Islanders (API) compared with 29 years for NHW patients (P = 0.0001). Five-year overall survival varied between 82% and 92% when diagnosed at the age of at least 40 years compared with less than 20 years (P0.0001), and from 85% to 95% in patients with distant vs local disease (P0.0001), respectively. Multivariate analysis demonstrated that age, race, and stage were independent predictors of mortality. Risk of death increased incrementally in patients diagnosed at 20 to 39 years of age (adjusted hazard ratio [aHR], 3.87; 95% confidence interval [CI], 1.69-8.86; P = 0.001) and at least 40 years of age (aHR, 7.18; 95% CI, 2.95-17.49; P0.0001) compared with 20 years or younger. Non-Hispanic black patients were the only racial group at higher risk of death compared with NHW patients (aHR, 1.86; 95% CI, 1.22-2.82; P0.004). Distant vs local disease added an additional risk of death (aHR, 2.43; 95% CI, 1.57-3.75; P0.0001) over that attributable to age at diagnosis and NHB race. Similar relationships to cancer-specific survival were also observed (P0.05).Most patients with choriocarcinoma have excellent prognosis. However, NHB patients and patients who are diagnosed at the age of at least 20 years or have distant stage have significantly worse mortality.

To investigate racial disparities in uterine carcinosarcoma (UCS) and ovarian carcinosarcoma (OCS) in Commission on Cancer®-accredited facilities.Non-Hispanic Black (NHB) and non-Hispanic White (NHW) women in the National Cancer Database diagnosed with stage I-IV UCS or OCS between 2004 and 2014 were eligible. Differences by disease site or race were compared using Chi-square test and multivariate Cox analysis.There were 2830 NHBs and 7366 NHWs with UCS, and 280 NHBs and 2586 NHWs with OCS. Diagnosis of UCS was more common in NHBs (11.5%) vs. NHWs (3.7%) and increased with age (P .0001). OCS diagnosis remained5% in both races and all ages. NHBs with UCS or OCS were more common in the South and more likely to have a comorbidity score ≥ 1, low neighborhood income and Medicaid or no insurance (P .0001). Diagnosis at stage II-IV was more common in NHBs than NHWs with UCS but not OCS. NHBs with both UCS and OCS were less likely to undergo surgery and to achieve no gross residual disease with surgery (P = .002). Risk of death in NHB vs. NHW patients with UCS was 1.38 after adjustment for demographic factors and dropped after sequential adjustment for comorbidity score, neighborhood income, insurance status, stage and treatment by 4%, 16%, 7%, 19% and 10%, respectively, leaving 43.5% of the racial disparity in survival unexplained. In contrast, risk of death in NHBs vs. NHWs with OCS was 1.19 after adjustment for demographic factors and became insignificant after adjustment for comorbidity. Race was an independent prognostic factor in UCS but not in OCS.Racial disparities exist in characteristics, treatment and survival in UCS and OCS with distinctions that merit additional research.

Prognostic and predictive biomarkers of disease and treatment outcome are needed to ensure optimal treatment of patients with triple-negative breast cancer (TNBC). In a mass spectrometry-based global proteomic study of 44 formalin-fixed, paraffin-embedded (FFPE) primary TNBC tumors and 10 corresponding metastases, we found that Cytochrome P450 reductase (CYPOR) expression correlated with patient outcome. The correlation between CYPOR expression and outcome was further evaluated in a Danish cohort of 113 TNBC patients using immunohistochemistry and publicly available gene expression data from two cohorts of TNBC and basal-like breast cancer patients, respectively (N = 249 and N = 580). A significant correlation between high CYPOR gene expression and shorter recurrence-free survival (RFS), but not overall survival, was found in the cohort of 249 TNBC patients (p = 0.018, HR = 1.77, 95% CI 1.1–2.85), and this correlation was recapitulated in a cohort of 580 basal-like breast cancer patients (p = 0.018, HR = 1.4, 95% CI 1.06–1.86). High CYPOR protein expression was also associated with shorter RFS in the cohort of 113 TNBC patients (p = 0.017, HR = 2.73, 95% CI 1.20–6.19), particularly those who were lymph node tumor-negative (p = 0.029, HR = 5.22). Multivariate Cox regression analysis identified CYPOR as an independent prognostic factor for shorter RFS in TNBC patients (p = 0.032, HR = 2.19, 95% CI 1.07–4.47). Together, these data suggest high expression of CYPOR as an independent prognostic biomarker of shorter RFS, which could be used to identify patients who should receive more extensive adjuvant treatment and more aggressive surveillance.

Rheumatoid arthritis is an inflammatory disease of the synovial joints that affects ~1% of the human population, with severe distress due to progressive joint inflammation and deformation. When addressing the links between specific components of the apoptotic cell clearance machinery and human disease, we noted a correlation between single nucleotide polymorphisms (SNPs) in ELMO1, DOCK2, and RAC1 genes and rheumatoid arthritis. ELMO1 is a cytoplasmic adapter protein that associates with DOCK2 and RAC1 to promote cytoskeletal reorganization needed for apoptotic cell uptake by phagocytes. We initially hypothesized that, since ELMO1 is linked to apoptotic cell clearance, loss of ELMO1 would lead to increased inflammation in arthritis. Contrary to the accumulation of apoptotic cells and greater disease severity that we predicted, we observed significantly reduced joint inflammation in two models of arthritis in mice lacking ELMO1. Using genetic and cell biological approaches in vivo and ex vivo, we determined that ELMO1 deficiency significantly reduces neutrophil recruitment to inflamed joints, but does not result in general inhibition of inflammatory responses. Through proteomic analyses, we find that ELMO1 protein associates with cellular receptors that contribute to neutrophil function in arthritis, and regulates C5a and LTB4 receptor-mediated activation and early neutrophil recruitment to the joints. Neutrophil-specific transcriptomics show that ELMO1 modulates neutrophil-specific gene expression that includes genes with known linkages to human arthritis. Finally, neutrophils from the peripheral blood of human donors that carry the SNP in ELMO1 associated with arthritis display increased migratory capacity, whereas ELMO1 knockdown reduces human neutrophil migration to LTB4. These data identify key ‘non-canonical’ roles for engulfment machinery components in arthritis, and ELMO1 as an important regulator of specific neutrophil receptors and signaling linked to arthritis.

Previously we have shown in endometrial cells that progesterone (P4) and calcitriol (CAL, 1,25(OH)2D3) synergistically promote apoptosis and that progestins induce expression of the vitamin D receptor. In the current study we examined the progestin/vitamin D combination in ovarian cells and searched for other progestin-related effects on vitamin D metabolism that may underlie the novel interaction between progestins and vitamin D, including whether progestins inhibit CYP24A1, the enzyme that renders CAL inactive.We investigated the impact of P4 on CAL-induced CYP24A1 expression in cancer cell lines expressing progesterone receptors (PRs), [OVCAR-5, OVCAR-3-PGR (PR-transfected OVCAR-3 ovarian line), and T47D-WT, T47D-A and T47D-B (breast lines expressing PRs or individual PR isoforms)] or lines that do not express PRs (OVCAR-3 and T47D-Y). We examined CYP24A1 expression using RT-PCR and western blotting, and apoptosis by TUNEL. We also investigated P4 inhibition of Cyp24a1 in ovaries from CAL-treated mice.CAL treatment induced CYP24A1 expression. When co-treated with P4, cell lines expressing PRs showed marked inhibition of CYP24A1 expression (p0.001), along with increased apoptosis (p0.01); cells not expressing PRs did not. Mouse ovaries showed a significant reduction in CAL-induced Cyp24a1 mRNA (p0.001) and protein (p0.01) in response to P4.We show for the first time that progestins and vitamin D synergistically reduce cell viability and induce apoptosis in ovarian cells and that progestins PR-dependently inhibit CAL-induced CYP24A1, thus extending CAL activity. The combination of progestins and vitamin D deserves further consideration as a strategy for inhibiting ovarian carcinogenesis.

We describe a dynamic phosphorylation on serine-1940 of the catalytic subunit of human Pol ε, POLE1, following DNA damage. We also describe novel interactions between POLE1 and the iron-sulfur cluster assembly complex CIA proteins CIAO1 and MMS19. We show that serine-1940 is essential for the interaction between POLE1 and MMS19, but not POLE1 and CIAO1. No defect in either proliferation or survival was identified when POLE1 serine-1940 was mutated to alanine in human cells, even following treatment with DNA damaging agents. We conclude that serine-1940 phosphorylation and the interaction between serine-1940 and MMS19 are not essential functions in the C terminal domain of the catalytic subunit of DNA polymerase ε.

SUMMARY The diversity and heterogeneity within high-grade serous ovarian cancer (HGSC), which is the most lethal gynecologic malignancy, is not well understood. Here, we perform comprehensive multi-platform omics analyses, including integrated analysis, and immune monitoring on primary and metastatic sites from highly clinically annotated HGSC samples based on a laparoscopic triage algorithm from patients who underwent complete gross resection (R0) or received neoadjuvant chemotherapy (NACT) with excellent or poor response. We identify significant distinct molecular abnormalities and cellular changes and immune cell repertoire alterations between the groups, including a higher rate of NF1 copy number loss, and reduced chromothripsis-like patterns, higher levels of strong-binding neoantigens, and a higher number of infiltrated T cells in the R0 versus the NACT groups., Graphical Abstract, In Brief High-grade serous ovarian cancer (HGSC) patients with no gross residual disease (R0) after primary surgery have the greatest improvement in clinical outcomes. A deep understanding of molecular and cellular heterogeneity of HGSC is lacking. Findings by Lee et al. highlight major molecular and cellular differences between clinically defined subgroups of HGSC.

ATR and ATM are DNA damage signaling kinases that phosphorylate several thousand substrates. ATR kinase activity is increased at damaged replication forks and resected DNA double-strand breaks (DSBs). ATM kinase activity is increased at DSBs. ATM has been widely studied since ataxia telangiectasia individuals who express no ATM protein are the most radiosensitive patients identified. Since ATM is not an essential protein, it is widely believed that ATM kinase inhibitors will be well-tolerated in the clinic. ATR has been widely studied, but advances have been complicated by the finding that ATR is an essential protein and it is widely believed that ATR kinase inhibitors will be toxic in the clinic. We describe AZD6738, an orally active and bioavailable ATR kinase inhibitor. AZD6738 induces cell death and senescence in non-small cell lung cancer (NSCLC) cell lines. AZD6738 potentiates the cytotoxicity of cisplatin and gemcitabine in NSCLC cell lines with intact ATM kinase signaling, and potently synergizes with cisplatin in ATM-deficient NSCLC cells. In contrast to expectations, daily administration of AZD6738 and ATR kinase inhibition for 14 consecutive days is tolerated in mice and enhances the therapeutic efficacy of cisplatin in xenograft models. Remarkably, the combination of cisplatin and AZD6738 resolves ATM-deficient lung cancer xenografts.

Functional loss of expression of breast cancer susceptibility gene 1(BRCA1) has been implicated in genomic instability and cancer progression. There is emerging evidence that BRCA1 gene product (BRCA1) also plays a role in cancer cell migration. We performed a quantitative proteomics study of EOC patient tumor tissues and identified changes in expression of several key regulators of actin cytoskeleton/cell adhesion and cell migration (CAPN1, 14-3-3, CAPG, PFN1, SPTBN1, CFN1) associated with loss of BRCA1 function. Gene expression analyses demonstrate that several of these proteomic hits are differentially expressed between early and advanced stage EOC thus suggesting clinical relevance of these proteins to disease progression. By immunohistochemistry of ovarian tumors with BRCA1(+/+) and BRCA1(null) status, we further verified our proteomic-based finding of elevated PFN1 expression associated with BRCA1 deficiency. Finally, we established a causal link between PFN1 and BRCA1-induced changes in cell migration thus uncovering a novel mechanistic basis for BRCA1-dependent regulation of ovarian cancer cell migration. Overall, findings of this study open up multiple avenues by which BRCA1 can potentially regulate migration and metastatic phenotype of EOC cells.

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.

Significant reductions in gynecologic (GYN) cancer mortality and morbidity require treatments that prevent and reverse resistance to chemotherapy and radiation. The objective of this study was to determine if pharmacologic inhibition of key DNA damage response kinases in GYN cancers would enhance cell killing by platinum-based chemotherapy and radiation.A panel of human ovarian, endometrial and cervical cancer cell lines were treated with platinum drugs or ionizing radiation (IR) along with small molecule pharmacological kinase inhibitors of Ataxia telangiectasia mutated (ATM) and ATM and Rad-3-related (ATR).Pharmacologic inhibition of ATR significantly enhanced platinum drug response in all GYN cancer cell lines tested, whereas inhibition of ATM did not enhance the response to platinum drugs. Co-inhibition of ATM and ATR did not enhance platinum kill beyond that observed by inhibition of ATR alone. By contrast, inhibiting either ATR or ATM enhanced the response to IR in all GYN cancer cells, with further enhancement achieved with co-inhibition.These studies highlight actionable mechanisms operative in GYN cancer cells with potential to maximize response of platinum agents and radiation in newly diagnosed as well as recurrent gynecologic cancers.

While mycosis fungoides (MF) is typically an indolent malignancy, it may infrequently undertake an aggressive course. We used proteomic analyses to identify a biomarker of the aggressive course of MF. Results of this investigation demonstrated that PARP-1, heat shock protein family A (Hsp70) member 1 like (HSAP1L), Hsp70 member 1A (HSPA1A), ATP-depending RNA-helicase (DDX17), and the α isoform of lamina-associated polypeptide 2 (TMPO) had higher expression in aggressive disease versus non-aggressive. Moreover, PARP-1 was overexpressed in patients with early stage of MF who developed later an aggressive disease. PARP-1 was evaluated as a new target for therapy, demonstrating the selective dose-dependent cytotoxic effect of PARP inhibitors on Sézary cells in comparison with non-malignant lymphocytes. In conclusion, we believe that PARP-1 may serve not only as a biomarker at initial biopsies for a disease that may become aggressive but also as a new therapeutic target of advanced MF and Sézary syndrome.

ATR kinase activity slows replication forks and prevents origin firing in damaged cells. Here we describe proteomic analyses that identified mechanisms through which ATR kinase inhibitors induce unscheduled origin firing in undamaged cells. ATR-Chk1 inhibitor-induced origin firing is mediated by Cdc7 kinase through previously undescribed phosphorylations on GINS that induce an association between GINS and And-1. ATR-Chk1 inhibitor-induced origin firing is blocked by prior exposure to DNA damaging agents showing that the prevention of origin firing does not require ongoing ATR activity. In contrast, ATR-Chk1 inhibitor-induced origins generate additional replication forks that are targeted by subsequent exposure to DNA damaging agents. Thus, the sequence of administration of an ATR kinase inhibitor and a DNA damaging agent impacts the DNA damage induced by the combination. Our experiments identify competing ATR and Cdc7 kinase-dependent mechanisms at replication origins in human cells., ATR kinase activity is essential for slowing replication forks and preventing DNA replication in cells with DNA damage. Here the authors show that ATR inhibition leads to Cdc7 phosphorylation of GINS, leading to origin firing.

INTRODUCTION. Although black patients with endometrial cancer (EC) have worse survival compared with white patients, the interaction between age/race has not been examined. The primary objective was to evaluate the impact of age at diagnosis on racial disparities in disease presentation and outcome in EC. METHODS. We evaluated women diagnosed with EC between 1991 and 2010 from the Surveillance, Epidemiology, and End Results. Mutation status for TP53 or PTEN, or with the aggressive integrative, transcript-based, or somatic copy number alteration-based molecular subtype were acquired from the Cancer Genome Atlas. Logistic regression model was used to estimate the interaction between age and race on histology. Cox regression model was used to estimate the interaction between age and race on survival. RESULTS. 78,184 white and 8518 black patients with EC were analyzed. Median age at diagnosis was 3-years younger for black vs. white patients with serous cancer and carcinosarcoma (P < 0.0001). The increased presentation of non-endometrioid histology with age was larger in black vs. white patients (P < 0.0001). The racial disparity in survival and cancer-related mortality was more prevalent in black vs. white patients, and in younger vs. older patients (P < 0.0001). Mutations in TP53, PTEN and the three aggressive molecular subtypes each varied by race, age and histology. CONCLUSIONS. Aggressive histology and molecular features were more common in black patients and older age, with greater impact of age on poor tumor characteristics in black vs. white patients. Racial disparities in outcome were larger in younger patients. Intervention at early ages may mitigate racial disparities in EC.

Selected reaction monitoring (SRM) is used in molecular profiling to detect and quantify specific known proteins in complex mixtures. Using isotope dilution (Barnidge et al., Anal Chem 75(3):445-451, 2003) methodologies, peptides can be quantified without the need for an antibody-based method. Selected reaction monitoring assays employ electrospray ionization mass spectrometry (ESI-MS) followed by two stages of mass selection: a first stage where the mass of the peptide ion is selected and, after fragmentation by collision-induced dissociation (CID), a second stage (tandem MS) where either a single (e.g., SRM) or multiple (multiple reaction monitoring, MRM) specific peptide fragment ions are transmitted for detection. The MRM experiment is accomplished by specifying the parent masses of the selected endogenous and isotope-labeled peptides for MS/MS fragmentation and then monitoring fragment ions of interest, using their intensities/abundances and relative ratios to quantify the parent protein of interest. In this example protocol, we will utilize isotope dilution MRM-MS to quantify in absolute terms the total levels of the protein of interest, ataxia telangiectasia mutated (ATM) serine/threonine protein kinase. Ataxia telangiectasia mutated (ATM) phosphorylates several key proteins that initiate activation of the DNA damage checkpoint leading to cell cycle arrest.

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.

Purpose: The EGF receptor (EGFR) and COX2 pathways are upregulated in head and neck squamous cell carcinoma (HNSCC). Preclinical models indicate synergistic antitumor activity from dual blockade. We conducted a randomized, double-blind, placebo-controlled window trial of erlotinib, an EGFR inhibitor; erlotinib plus sulindac, a nonselective COX inhibitor; versus placebo. Experimental Design: Patients with untreated, operable stage II-IVb HNSCC were randomized 5:5:3 to erlotinib, erlotinib–sulindac, or placebo. Tumor specimens were collected before and after seven to 14 days of treatment. The primary endpoint was change in Ki67 proliferation index. We hypothesized an ordering effect in Ki67 reduction: erlotinib–sulindac > erlotinib > placebo. We evaluated tissue microarrays by immunohistochemistry for pharmacodynamic modulation of EGFR and COX2 signaling intermediates. Results: From 2005–2009, 47 patients were randomized for the target 39 evaluable patients. Thirty-four tumor pairs were of sufficient quality to assess biomarker modulation. Ki67 was significantly decreased by erlotinib or erlotinib–sulindac (omnibus comparison, two-sided Kruskal–Wallis, P = 0.04). Wilcoxon pairwise contrasts confirmed greater Ki67 effect in both erlotinib groups (erlotinib–sulindac vs. placebo, P = 0.043; erlotinib vs. placebo, P = 0.027). There was a significant trend in ordering of Ki67 reduction: erlotinib–sulindac > erlotinib > placebo (two-sided exact Jonckheere–Terpstra, P = 0.0185). Low baseline pSrc correlated with greater Ki67 reduction (R2 = 0.312, P = 0.024). Conclusions: Brief treatment with erlotinib significantly decreased proliferation in HNSCC, with additive effect from sulindac. Efficacy studies of dual EGFR–COX inhibition are justified. pSrc is a potential resistance biomarker for anti-EGFR therapy, and warrants investigation as a molecular target. Clin Cancer Res; 20(12); 3289–98. ©2014 AACR.

Endometrial cancer is the most common gynecological cancer in the United States. However, no early detection test exists for asymptomatic women at average risk for endometrial cancer.We sought to identify early detection biomarkers for endometrial cancer using prediagnostic serum.We performed a nested case-control study of postmenopausal women in the Prostate, Lung, Colorectal, and Ovarian cancer screening trial (n = 78,216), including 112 incident endometrial cancer cases and 112 controls. Prediagnostic serum was immunodepleted of high-abundance proteins and digested with sequencing grade porcine trypsin via pressure cycling technology. Quantitative proteomics and phosphoproteomics was performed using high-resolution liquid chromatography-tandem mass spectrometry and highly multiplexed isobaric mass tag combined with basic reversed-phase liquid chromatography. A set of proteins able to predict cancer status was identified with an integrated score assessed by receiver-operator curve analysis.Mean time from blood draw to endometrial cancer diagnosis was 3.5 years (SD, 1.9 years). There were 47 differentially abundant proteins between cases and controls (P.05). Protein alterations with high predictive potential were selected by regression analysis and compiled into an aggregate score to determine the ability to predict endometrial cancer. An integrated risk score of 6 proteins was directly related to disease incidence in cases with blood draw ≤2 years,2 years to ≤5 years or5 years prior to cancer diagnosis. The integrated score distinguished cases from controls with an area under the curve of 0.80 (95% confidence interval, 0.72-0.88).An integrated score of 6 proteins using prediagnostic serum from the Prostate, Lung, Colorectal, and Ovarian cancer screening trial distinguishes postmenopausal endometrial cancer cases from controls. Validation is needed to evaluate whether this test can improve prediction or detection of endometrial cancer among postmenopausal women.

Background: The majority of patients diagnosed with advanced epithelial ovarian carcinoma (EOC) relapse with resistant disease, and there are no biomarkers that possess clinical utility to identify or monitor these patients. This study aimed to identify secreted proteins (‘secretome') collected from human EOC cell lines that differ in their inherent platinum sensitivity. Methods: Secreted proteins collected from conditioned medium from ovarian cancer cell lines that vary in their sensitivity to cisplatin were digested with trypsin and analysed by liquid chromatography-tandem mass spectrometry for peptide identification. Results: Of the 1688 proteins identified, 16 possessed significant differential abundances (P

Human studies suggest that progesterone and calcitriol may prove beneficial in preventing or inhibiting oncogenesis, but the underlying mechanism is not fully understood. The current study investigates the effects of progesterone, calcitriol, and their combination on immortalized human endometrial epithelial cells and endometrial cancer cells and identifies their targets of action. Combination treatment with both agents enhanced vitamin D receptor expression and inhibited cell proliferation through caspase-3 activation and induction of G0–G1 cell-cycle arrest with associated downregulation of cyclins D1 and D3 and p27 induction. We used mass spectrometry–based proteomics to measure protein abundance differences between calcitriol-, progesterone-, or combination-exposed endometrial cells. A total of 117 proteins showed differential expression among these three treatments. Four proteins were then selected for validation studies: histone H1.4 (HIST1H1E), histidine triad nucleotide-binding protein 2 (HINT2), IFN-induced, double-stranded RNA-activated protein kinase (EIF2AK2), and Bcl-2–associated X protein (BAX). Abundance levels of selected candidates were low in endometrial cancer cell lines versus the immortalized endometrial epithelial cell line. All four proteins displayed elevated expression in cancer cells upon exposure to calcitriol, progesterone, or the combination. Further BAX analysis through gain- or loss-of-function experiments revealed that upregulation of BAX decreased cell proliferation by changing the BAX:BCL-2 ratio. Knockdown of BAX attenuated progesterone- and calcitriol-induced cell growth inhibition. Our results showed that progesterone and calcitriol upregulate the expression of BAX along with other apoptosis-related proteins, which induce inhibition of endometrial cancer cell growth by apoptosis and cell-cycle arrest. Cancer Prev Res; 6(7); 731–43. ©2013 AACR.

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.

Recent studies have suggested that changes in serum phosphate levels influence pathological states associated with aging such as cancer, bone metabolism, and cardiovascular function, even in individuals with normal renal function. The causes are only beginning to be elucidated but are likely a combination of endocrine, paracrine, autocrine, and cell autonomous effects. We have used an integrated quantitative biology approach, combining transcriptomics and proteomics to define a multi-phase, extracellular phosphate-induced, signaling network in pre-osteoblasts as well as primary human and mouse mesenchymal stromal cells. We identified a rapid mitogenic response stimulated by elevated phosphate that results in the induction of immediate early genes including c-fos. The mechanism of activation requires FGF receptor signaling followed by stimulation of N-ras and activation of AP-1 and serum response elements. A distinct long-term response also requires FGF receptor signaling and results in N-ras activation and expression of genes and secretion of proteins involved in matrix regulation, calcification, and angiogenesis. The late response is synergistically enhanced by addition of FGF23 peptide. The intermediate phase results in increased oxidative phosphorylation and ATP production and is necessary for the late response providing a functional link between the phases. Collectively, the results define elevated phosphate, as a mitogen and define specific mechanisms by which phosphate stimulates proliferation and matrix regulation. Our approach provides a comprehensive understanding of the cellular response to elevated extracellular phosphate, functionally connecting temporally coordinated signaling, transcriptional, and metabolic events with changes in long-term cell behavior.

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.

// Doreen O. Jackson 1 , Kevin Byrd 2,3 , Timothy J. Vreeland 4 , Diane F. Hale 1 , Garth S. Herbert 1 , Julia M. Greene 1 , Erika J. Schneble 1 , John S. Berry 1 , Alfred F. Trappey 1 , Guy Travis Clifton 5 , Mark O. Hardin 6 , Jonathan Martin 7 , John C. Elkas 8,9 , Thomas P. Conrads 2,3,8,10 , Kathleen M. Darcy 2,3 , Chad A. Hamilton 2,3 , George L. Maxwell 2,3,8,10 and George E. Peoples 7 1 Department of Surgery, San Antonio Military Medical Center, San Antonio, TX, USA 2 National Capital Consortium Fellowship in Gynecologic Oncology, Walter Reed National Military Medical Center Bethesda, MD, USA 3 Gynecologic Cancer Center of Excellence, Annandale, VA, USA 4 Department of Surgery, Womack Army Medical Center, Fayetteville, NC, USA 5 Department of Surgical Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA 6 Department of Surgery, Madigan Army Medical Center, Tacoma, WA, USA 7 Cancer Vaccine Development Program, San Antonio, TX, USA 8 Department of Obstetrics and Gynecology, Inova Fairfax Hospital Annandale, VA, USA 9 Mid-Atlantic Gynecologic Oncology and Pelvic Surgical Associates, Annandale, VA, USA 10 Inova Schar Cancer Institute, Inova Health System, Annandale, VA, USA Correspondence to: Doreen O. Jackson, email: // Keywords : cancer, ovarian, endometrial, folate binding protein, immunotherapy Received : July 01, 2016 Accepted : October 19, 2016 Published : November 11, 2016 Abstract BACKGROUND: Folate binding protein(FBP) is an immunogenic protein over-expressed in endometrial(EC) and ovarian cancer(OC). We are conducting a phase I/IIa trial of E39 (GALE 301)+GM-CSF, an HLA-A2-restricted, FBP-derived peptide vaccine to prevent recurrences in disease-free EC and OC patients. This interim analysis summarizes toxicity, immunologic responses, and clinical outcomes to date. METHODS: HLA-A2+ patients were vaccinated(VG), and HLA-A2- or -A2+ patients were followed as controls(CG). Six monthly intradermal inoculations of E39+250mcg GM-CSF were administered to VG. Demographic, safety, immunologic, and recurrence rate(RR) data were collected and evaluated. RESULTS: This trial enrolled 51 patients; 29 in the VG and 22 in the CG. Fifteen patients received 1000mcg E39, and 14 received

// Liza C. Villaruz 1, * , Helen Jones 2, * , Sanja Dacic 3 , Shira Abberbock 1 , Brenda F. Kurland 1, 4 , Laura P. Stabile 5 , Jill M. Siegfried 6 , Thomas P. Conrads 7 , Neil R. Smith 2 , Mark J. O’Connor 2 , Andrew J. Pierce 2 , Christopher J. Bakkenist 5, 8 1 University of Pittsburgh Cancer Institute, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA 2 Astrazeneca, Cambridge, United Kingdom 3 Department of Pathology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA 4 Department of Biostatistics, University of Pittsburgh School of Public Health, Pittsburgh, PA, USA 5 Department of Pharmacology and Chemical Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA 6 Department of Pharmacology, University of Minnesota, Minneapolis, MN, USA 7 Inova Schar Cancer Institute, Inova Center for Personalized Health, Falls Church, VA, USA 8 Department of Radiation Oncology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA * Co-first authors Correspondence to: Christopher J. Bakkenist, email: bakkenistcj@upmc.edu Keywords: ataxia telangiectasia mutated (ATM), ATM and Rad-3-related (ATR), lung adenocarcinoma, non-small cell lung cancer (NSCLC) Received: March 21, 2016 Accepted: April 28, 2016 Published: June 01, 2016 ABSTRACT Lung cancer is the leading cause of cancer-related mortality in the USA and worldwide, and of the estimated 1.2 million new cases of lung cancer diagnosed every year, over 30% are lung adenocarcinomas. The backbone of 1 st -line systemic therapy in the metastatic setting, in the absence of an actionable oncogenic driver, is platinum-based chemotherapy. ATM and ATR are DNA damage signaling kinases activated at DNA double-strand breaks (DSBs) and stalled and collapsed replication forks, respectively. ATM protein is lost in a number of cancer cell lines and ATR kinase inhibitors synergize with cisplatin to resolve xenograft models of ATM-deficient lung cancer. We therefore sought to determine the frequency of ATM loss in a tissue microarray (TMA) of lung adenocarcinoma. Here we report the validation of a commercial antibody (ab32420) for the identification of ATM by immunohistochemistry and estimate that 61 of 147 (41%, 95% CI 34%-50%) cases of lung adenocarcinoma are negative for ATM protein expression. As a positive control for ATM staining, nuclear ATM protein was identified in stroma and immune infiltrate in all evaluable cases. ATM loss in lung adenocarcinoma was not associated with overall survival. However, our preclinical findings in ATM-deficient cell lines suggest that ATM could be a predictive biomarker for synergy of an ATR kinase inhibitor with standard-of-care cisplatin. This could improve clinical outcome in 100,000’s of patients with ATM-deficient lung adenocarcinoma every year.

The Cancer Moonshot Program has been launched and represents a potentially paradigm-shifting initiative with the goal to implement a focused national effort to double the rate of progress against cancer. The placement of precision medicine, immunotherapy, genomics, and combination therapies was placed at the central nexus of this initiative. Although we are extremely enthusiastic about the goals of the program, it is time we meet this revolutionary project with equally bold and cutting-edge ideas: it is time we move firmly into the postgenome era and provide the necessary resources to propel and seize on innovative recent gains in the field of proteomics required for it to stand on equal footing in this narrative as a combined, synergistic engine for molecular profiling. After all, although the genome is the information archive, it is the proteins that actually do the work of the cell and represent the structural cellular machinery. It is the proteins that comprise most of the biomarkers that are measured to detect cancers, constitute the antigens that drive immune response and inter- and intracellular communications, and it is the proteins that are the drug targets for nearly every targeted therapy that is being evaluated in cancer trials today. We believe that a combined systems biology view of the tumor microenvironment that orients cancer studies back to the functional proteome, phosphoproteome, and biochemistry of the cell will be essential to deliver on the promise of the Cancer Moonshot Program. Clin Cancer Res; 22(18); 4556–8. ©2016 AACR.