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PURPOSE The status of p53 in a tumor can be inferred by next-generation sequencing (NGS) or by immunohistochemistry (IHC). We examined the association between p53 IHC and sequence and whether p53 IHC alone, or integrated with TP53 NGS, predicts the outcome. METHODS From GOG-86P, a randomized phase II study of chemotherapy combined with either bevacizumab or temsirolimus in advanced endometrial cancer, 213 cases had p53 protein expression data measured by IHC and TP53 NGS data. An analysis was designed to integrate p53 expression by IHC with the presence or absence of a TP53 mutation. These variables were further correlated with progression-free survival (PFS) and overall survival (OS) in the chemotherapy plus bevacizumab arms versus the chemotherapy plus temsirolimus arm. RESULTS In the analysis of p53 IHC, the most striking treatment effect favoring bevacizumab was in cases where p53 was overexpressed (PFS hazard ratio [HR]: 0.46, 95% CI, 0.26 to 0.88; OS HR: 0.31, 95% CI, 0.16 to 0.62). On integrated analysis, patients with TP53 missense mutations and p53 protein overexpression had a similar treatment effect on PFS (HR: 0.41, 95% CI, 0.22 to 0.83) and OS (HR: 0.28, 95% CI, 0.14 to 0.59) favoring bevacizumab plus chemotherapy relative to temsirolimus plus chemotherapy. Concordance between TP53 NGS and p53 IHC was 88%. Concordance was 92% when cases with TP53 mutations and POLE mutations or mismatch repair deficiency were removed. CONCLUSION IHC for p53 alone or when integrated with sequencing for TP53 identifies a specific, high-risk tumor genotype/phenotype for which bevacizumab is particularly beneficial in improving outcomes when combined with chemotherapy.

The creation of well-understood structures using spectral hole burning is an important task in the use of technologies based on rare-earth ion-doped crystals. We apply a series of different techniques to model and improve the frequency dependent population change in the atomic level structure of thulium yttrium gallium garnet (Tm:YGG). In particular we demonstrate that, at zero applied magnetic field, numerical solutions to frequency-dependent three-level rate equations show good agreement with spectral hole-burning results. This allows us to predict spectral structures given a specific hole-burning sequence, the underpinning spectroscopic material properties, and the relevant laser parameters. This enables us to largely eliminate power-dependent hole broadening through the use of adiabatic hole-burning pulses. Although this system of rate equations shows good agreement at zero field, the addition of a magnetic field results in unexpected spectral diffusion proportional to the induced Tm ion magnetic-dipole moment and average magnetic-field strength, which, through the quadratic Zeeman effect, dominates the optical spectrum over long timescales. Our results allow optimization of the preparation process for spectral structures in a large variety of rare-earth ion-doped materials for quantum memories and other applications.

Most endometrial cancers express the hormone receptor estrogen receptor alpha (ER) and are driven by excess estrogen signaling. However, evaluation of the estrogen response in endometrial cancer cells has been limited by the availability of hormonally responsive in vitro models, with one cell line, Ishikawa, being used in most studies. Here, we describe a novel, adherent endometrioid endometrial cancer (EEC) cell line model, HCI-EC-23. We show that HCI-EC-23 retains ER expression and that ER functionally responds to estrogen induction over a range of passages. We also demonstrate that this cell line retains paradoxical activation of ER by tamoxifen, which is also observed in Ishikawa and is consistent with clinical data. The mutational landscape shows that HCI-EC-23 is mutated at many of the commonly altered genes in EEC, has relatively few copy-number alterations, and is microsatellite instable high (MSI-high). In vitro proliferation of HCI-EC-23 is strongly reduced upon combination estrogen and progesterone treatment. HCI-EC-23 exhibits strong estrogen dependence for tumor growth in vivo and tumor size is reduced by combination estrogen and progesterone treatment. Molecular characterization of estrogen induction in HCI-EC-23 revealed hundreds of estrogen-responsive genes that significantly overlapped with those regulated in Ishikawa. Analysis of ER genome binding identified similar patterns in HCI-EC-23 and Ishikawa, although ER exhibited more bound sites in Ishikawa. This study demonstrates that HCI-EC-23 is an estrogen- and progesterone-responsive cell line model that can be used to study the hormonal aspects of endometrial cancer.

Most endometrial cancers express the hormone receptor estrogen receptor alpha (ER) and are driven by excess estrogen signaling. However, evaluation of the estrogen response in endometrial cancer cells has been limited by the availability of hormonally responsivein vitromodels, with one cell line, Ishikawa, being used in most studies. Here, we describe a novel, adherent endometrioid endometrial cancer (EEC) cell line model, HCI-EC-23. We show that HCI-EC-23 retains ER expression and that ER functionally responds to estrogen induction over a range of passages. We also demonstrate that this cell line retains paradoxical activation of ER by tamoxifen, which is also observed in Ishikawa and is consistent with clinical data. The mutational landscape shows that HCI-EC-23 is mutated at many of the commonly altered genes in EEC, has relatively few copy-number alterations, and is microsatellite instable high (MSI-high).In vitroproliferation of HCI-EC-23 is strongly reduced upon combination estrogen and progesterone treatment. HCI-EC-23 exhibits strong estrogen dependence for tumor growthin vivoand tumor size is reduced by combination estrogen and progesterone treatment. Molecular characterization of estrogen induction in HCI-EC-23 revealed hundreds of estrogen-responsive genes that significantly overlapped with those regulated in Ishikawa. Analysis of ER genome binding identified similar patterns in HCI-EC-23 and Ishikawa, although ER exhibited more bound sites in Ishikawa. This study demonstrates that HCI-EC-23 is an estrogen- and progesterone-responsive cell line model that can be used to study the hormonal aspects of endometrial cancer.

Background Successfully combining targeted agents with chemotherapy is an important future goal for cancer therapy. However, an improvement in patient outcomes requires an enhanced understanding of the tumor biomarkers that predict for drug sensitivity. NRG Oncology/Gynecologic Oncology Group (GOG) Study GOG-86P was one of the first attempts to combine targeted agents (bevacizumab or temsirolimus) with chemotherapy in patients with advanced endometrial cancer. Herein we performed exploratory analyses to examine the relationship between mutations in TP53, the most commonly mutated gene in cancer, with outcomes on GOG-86P. Methods TP53 mutational status was determined and correlated with progression-free survival (PFS) and overall survival (OS) on GOG-86P. Results Mutations in TP53 were associated with improved PFS and OS for patients that received bevacizumab as compared to temsirolimus (PFS: HR 0.48, 95% CI 0.31, 0.75; OS: HR: 0.61, 95% CI 0.38, 0.98). By contrast, there was no statistically significant difference in PFS or OS between arms for cases with WT TP53. Conclusions This exploratory study suggests that combining chemotherapy with bevacizumab, but not temsirolimus, may enhance PFS and OS for patients whose tumors harbor mutant p53. These data set the stage for larger clinical studies evaluating the potential of TP53 mutational status as a biomarker to guide choice of treatment for endometrial cancer patients. Clintrials.gov : NCT00977574 .

Long optical storage times are an essential requirement to establish high-rate entanglement distribution over large distances using memory-based quantum repeaters. Rare earth ion-doped crystals are arguably well-suited candidates for building such quantum memories. Toward this end, we investigate the 795.32 nm 3H6 ↔ 3H4 transition of 1% thulium-doped yttrium gallium garnet crystal (Tm3+:Y3Ga5O12 : Tm3+:YGG). Most essentially, we find that the optical coherence time can reach 1.1 ms, and, using laser pulses, we demonstrate optical storage based on the atomic frequency comb (AFC) protocol up to 100 µs. In addition, we demonstrate multiplexed storage, including feed-forward selection, shifting, and filtering of spectral modes, as well as quantum state storage using members of non-classical photon pairs. Our results show that Tm:YGG can be a potential candidate for creating multiplexed quantum memories with long optical storage times.

Histone deacetylase (HDAC) inhibitors and proteasome inhibitors have been approved by the FDA for the treatment of multiple myeloma and lymphoma, respectively, but have not achieved similar activity as single agents in solid tumors. Preclinical studies have demonstrated the activity of the combination of an HDAC inhibitor and a proteasome inhibitor in a variety of tumor models. However, the mechanisms underlying sensitivity and resistance to this combination are not well-understood. This study explores the role of autophagy in adaptive resistance to dual HDAC and proteasome inhibition. Studies focus on ovarian and endometrial gynecologic cancers, two diseases with high mortality and a need for novel treatment approaches. We found that nanomolar concentrations of the proteasome inhibitor ixazomib and HDAC inhibitor romidepsin synergistically induce cell death in the majority of gynecologic cancer cells and patient-derived organoid (PDO) models created using endometrial and ovarian patient tumor tissue. However, some models were not sensitive to this combination, and mechanistic studies implicated autophagy as the main mediator of cell survival in the context of dual HDAC and proteasome inhibition. Whereas the combination of ixazomib and romidepsin reduces autophagy in sensitive gynecologic cancer models, autophagy is induced following drug treatment of resistant cells. Pharmacologic or genetic inhibition of autophagy in resistant cells reverses drug resistance as evidenced by an enhanced anti-tumor response both in vitro and in vivo. Taken together, our findings demonstrate a role for autophagic-mediated cell survival in proteasome inhibitor and HDAC inhibitor-resistant gynecologic cancer cells. These data reveal a new approach to overcome drug resistance by inhibiting the autophagy pathway.

In this work, we fabricate a multimode quantum memory out of a thuliumdoped crystal and demonstrate storage of laser pulses of up to 100 µsec. A significant step forward for creating quantum memories with long optical storage times.

Rare-earth-ion doped crystals are a promising platform for applications in microwave-to-optical transduction. Erbium is of special interest because of its telecom wavelength optical transitions. This work investigates the optical and electron-spin properties of Er${}^{3+}$:YVO${}_{4}$. An initial transduction of classical microwave signal using this material is demonstrated. The measured strong optical dipole moment and narrow ensemble inhomogeneities make Er${}^{3+}$:YVO${}_{4}$ a promising material for microwave-to-optical transduction.

Simple Summary In the conventional treatment of gynecologic malignancies, most patients receive similar ‘one-size-fits-all’ treatment. However, it is increasingly clear that standard therapies do not work in every patient, and it would be very helpful to have pretreatment predictive assays to provide more personalized regimens. In this study, we describe the routine, successful establishment of patient-derived organoid models (PDOs) of endometrial and ovarian cancer tissues from consenting patients and provide an example of how information from drug screening in PDOs may be a useful predictor of patient response to therapy. Abstract Developing reliable experimental models that can predict clinical response before treating the patient is a high priority in gynecologic cancer research, especially in advanced or recurrent endometrial and ovarian cancers. Patient-derived organoids (PDOs) represent such an opportunity. Herein, we describe our successful creation of 43 tumor organoid cultures and nine adjacent normal tissue organoid cultures derived from patients with endometrial or ovarian cancer. From an initial set of 45 tumor tissues and seven ascites fluid samples harvested at surgery, 83% grew as organoids. Drug sensitivity testing and organoid cell viability assays were performed in 19 PDOs, a process that was accomplished within seven days of obtaining the initial surgical tumor sample. Sufficient numbers of cells were obtained to facilitate testing of the most commonly used agents for ovarian and endometrial cancer. The models reflected a range of sensitivity to platinum-containing chemotherapy as well as other relevant agents. One PDO from a patient treated prior to surgery with neoadjuvant trastuzumab successfully predicted the patient’s postoperative chemotherapy and trastuzumab resistance. In addition, the PDO drug sensitivity assay identified alternative treatment options that are currently used in the second-line setting. Our findings suggest that PDOs could be used as a preclinical platform for personalized cancer therapy for gynecologic cancer patients.

Objectives: Endometrial cancer incidence and mortality are rising in the US. Disease recurrence has been shown to have a significant impact on mortality. However, to date, there are no accurate and validated prediction models that would discriminate which individual patients are likely to recur. Reliably predicting recurrence would be of benefit for treatment decisions following surgery. We present an integrated model constructed with comprehensive clinical, pathological and molecular features designed to discriminate risk of recurrence for patients with endometrioid endometrial adenocarcinoma. Subjects and methods: A cohort of endometrioid endometrial cancer patients treated at our institution was assembled. Clinical characteristics were extracted from patient charts. Primary tumors from these patients were obtained and total tissue RNA extracted for RNA sequencing. A prediction model was designed containing both clinical characteristics and molecular profiling of the tumors. The same analysis was carried out with data derived from The Cancer Genome Atlas for replication and external validation. Results: Prediction models derived from our institutional data predicted recurrence with high accuracy as evidenced by areas under the curve approaching 1. Similar trends were observed in the analysis of TCGA data. Further, a scoring system for risk of recurrence was devised that showed specificities as high as 81% and negative predictive value as high as 90%. Lastly, we identify specific molecular characteristics of patient tumors that may contribute to the process of disease recurrence. Conclusion: By constructing a comprehensive model, we are able to reliably predict recurrence in endometrioid endometrial cancer. We devised a clinically useful scoring system and thresholds to discriminate risk of recurrence. Finally, the data presented here open a window to understanding the mechanisms of recurrence in endometrial cancer.

Simple Summary Wnt has diverse regulatory roles at multiple cellular levels and numerous targeting points, and aberrant Wnt signaling has crucial roles in carcinogenesis, metastasis, cancer recurrence, and chemotherapy resistance; based on these facts, Wnt represents an appealing therapeutic target for cancer treatment. Although preclinical data supports a role for the Wnt signaling pathway in uterine carcinogenesis, this area remains understudied. In this review, we identify the functions of several oncogenes of the Wnt/β-catenin signaling pathway in tumorigenesis and address the translation approach with potent Wnt inhibitors that have already been established or are being investigated to target key components of the pathway. Further research is likely to expand the potential for both biomarker and cancer drug development. There is a scarcity of treatment choices for advanced and recurrent endometrial cancer; investigating the sophisticated connections of Wnt signaling networks in endometrial cancer could address the unmet need for new therapeutic targets. Abstract This review presents new findings on Wnt signaling in endometrial carcinoma and implications for possible future treatments. The Wnt proteins are essential mediators in cell signaling during vertebrate embryo development. Recent biochemical and genetic studies have provided significant insight into Wnt signaling, in particular in cell cycle regulation, inflammation, and cancer. The role of Wnt signaling is well established in gastrointestinal and breast cancers, but its function in gynecologic cancers, especially in endometrial cancers, has not been well elucidated. Development of a subset of endometrial carcinomas has been attributed to activation of the APC/β-catenin signaling pathway (due to β-catenin mutations) and downregulation of Wnt antagonists by epigenetic silencing. The Wnt pathway also appears to be linked to estrogen and progesterone, and new findings implicate it in mTOR and Hedgehog signaling. Therapeutic interference of Wnt signaling remains a significant challenge. Herein, we discuss the Wnt-activating mechanisms in endometrial cancer and review the current advances and challenges in drug discovery.

We characterize the optical coherence and energy-level properties of the 795-nm $^{3}\mathrm{H}_{6}$ to $^{3}\mathrm{H}_{4}$ transition of ${\mathrm{Tm}}^{3+}$ in a ${\mathrm{Ti}}^{4+}$:$\mathrm{Li}\mathrm{Nb}{\mathrm{O}}_{3}$ waveguide at temperatures as low as 0.65 K. Coherence properties are measured with varied temperature, magnetic field, optical excitation power and wavelength, and measurement timescale. We also investigate nuclear spin-induced hyperfine structure and population dynamics with varying magnetic field and laser excitation power. Except for accountable differences due to different ${\mathrm{Ti}}^{4+}$- and ${\mathrm{Tm}}^{3+}$-doping concentrations, we find that the properties of ${\mathrm{Tm}}^{3+}$:${\mathrm{Ti}}^{4+}$:$\mathrm{Li}\mathrm{Nb}{\mathrm{O}}_{3}$ produced by indiffusion doping are consistent with those of a bulk-doped ${\mathrm{Tm}}^{3+}$:$\mathrm{Li}\mathrm{Nb}{\mathrm{O}}_{3}$ crystal measured under similar conditions. Our results, which complement previous work in a narrower parameter space, support using rare-earth ions for integrated optical and quantum signal processing.

We characterize the magnetic properties for thulium ion energy levels in the Y$_3$Ga$_5$O$_{12}$ (Tm:YGG) lattice with the goal to improve decoherence and reduce line-width broadening caused by local host spins and crystal imperfections. More precisely, we measure hyperfine tensors for the lowest level of the, $^3$H$_6$, and excited, $^3$H$_4$, states using a combination of spectral hole burning, absorption spectroscopy, and optically detected nuclear magnetic resonance. By rotating the sample through a series of angles with an applied external magnetic field, we measure and analyze the orientation dependence of the Tm$^{3+}$ ion's spin-Hamiltonian. Using this spin-Hamiltonian, we propose a set of orientations to improve material properties that are important for light-matter interaction and quantum information applications. Our results yield several important external field directions: some to extend optical coherence times, another to improve spin inhomogeneous broadening, and yet another that maximizes mixing of the spin states for specific sets of ions, which allows improving optical pumping and creation of lambda systems in this material.

We argue that long optical storage times are required to establish entanglement at high rates over large distances using memory-based quantum repeaters. Triggered by this conclusion, we investigate the 795.325 nm^{3} H_{6}↔^{3}H_{4} transition of Tm:Y_{3}Ga_{5}O_{12} (Tm:YGG). Most importantly, we find that the optical coherence time can reach 1.1 ms, and, using laser pulses, we demonstrate optical storage based on the atomic frequency comb protocol during up to 100 μs as well as a memory decay time T_{m} of 13.1 μs. Possibilities of how to narrow the gap between the measured value of T_{m} and its maximum of 275 μs are discussed. In addition, we demonstrate multiplexed storage, including with feed-forward selection, shifting and filtering of spectral modes, as well as quantum state storage using members of nonclassical photon pairs. Our results show the potential of Tm:YGG for creating multiplexed quantum memories with long optical storage times, and open the path to repeater-based quantum networks with high entanglement distribution rates.

Objectives: Antiangiogenics have become a cornerstone of treatment in advanced stage and recurrent high grade serous ovarian cancer. Both cediranib (ICON6) and bevacizumab (ICON7, GOG218) have demonstrated benefit as a maintenance treatment for ovarian cancer patients in clinical trials. Investigators have also explored their role in advanced stage and recurrent endometrial carcinoma (MITO-END 2, Rose 2017, Aghajanian 2011). There have been no head to head trials in gynecologic cancers comparing cediranib to bevacizumab, although in colorectal carcinoma, phase III data suggests equivalent efficacy (HORIZON III). We hypothesize that these drugs, although both categorized as anti-angiogenics, induce unique cellular responses and sought to explore the varying impact of each agent on cell signaling using established serous cell models. Methods: CAOV3 ovarian cancer cells and Hec50 endometrial cancer cells were exposed to 1mM bevacizumab or cediranib for 24 hours. Cell lysates were analyzed in duplicate using an antibody-based phosphoproteomic array of 875 phosphoproteins supplied by Kinexus Bioinformatics (Vancouver, BC). Priority signaling effects were defined for each drug and in each cell line as those with a significant (>75%) change from control with an acceptable sum of error ( Results: Bevacizumab yielded 13 total leads, and cediranib yielded 9 in CAOV3 ovarian cancer cells (Table 1). Both agents significantly enhanced signaling events on the following epitopes: ABl, EZH2, HGK (2 sites), and IGF1R, indicating that these may be some of the core cellular effects of anti-angiogenic agents. Interestingly, all common leads induced by both agents in CAOV3 cells represent potential pro-oncogenic effects. In Hec50 cells (Table 1), both cediranib and bevacizumab treatment induced a unique set of phosphoproteins compared to the effects of these agents in CAOV3 cells, yet common findings were the phosphorylation of IGFR1 by bevacizumab and the activation of alternative MAP kinases in both cell lines. Download : Download high-res image (128KB) Download : Download full-size image Conclusions: Angiogenesis plays a crucial role in tumor development and metastasis. Blocking angiogenesis using monoclonal antibodies such as bevacizumab or tyrosine kinase inhibitors such as cediranib is a strategy that is proving to be helpful in the care of patients with gynecologic malignancies. These molecular agents have both on-target, therapeutic effects but also may induce pathways of resistance. In these comprehensive phosphoproteomic mapping studies, we noted the activation of signaling pathways which were cell and drug specific. One consistent finding was the enhanced phosphorylation of IGFR1 and alternative MAP kinases that may portend eventual drug resistance.

Nearly a third of patients with high-grade serous ovarian cancer (HGSC) do not respond to initial therapy and have an overall poor prognosis. However, there are no validated tools that accurately predict which patients will not respond. Our objective is to create and validate accurate models of prediction for treatment response in HGSC. This is a retrospective case–control study that integrates comprehensive clinical and genomic data from 88 patients with HGSC from a single institution. Responders were those patients with a progression-free survival of at least 6 months after treatment. Only patients with complete clinical information and frozen specimen at surgery were included. Gene, miRNA, exon, and long non-coding RNA (lncRNA) expression, gene copy number, genomic variation, and fusion-gene determination were extracted from RNA-sequencing data. DNA methylation analysis was performed. Initial selection of informative variables was performed with univariate ANOVA with cross-validation. Significant variables (p

As an active material with favorable linear and nonlinear optical properties, thin-film lithium niobate has demonstrated its potential in integrated photonics. Integration with rare-earth ions, which are promising candidates for quantum memories and transducers, will enrich the system with new applications in quantum information processing. Here, we investigate the optical properties at 1.5 micron wavelengths of rare-earth ions (Er$^{3+}$) implanted in thin-film lithium niobate waveguides and micro-ring resonators. Optical quality factors near a million after post annealing show that ion implantation damage can be successfully repaired. The transition linewidth and fluorescence lifetime of erbium ions are characterized, revealing values comparable to bulk-doped crystals. The ion-cavity coupling is observed through a Purcell enhanced fluorescence, from which a Purcell factor of ~3.8 is extracted. This platform is compatible with top-down lithography processes and leads to a scalable path for controlling spin-photon interfaces in photonic circuits.

Uterine serous carcinoma, one of the most aggressive types of endometrial cancer, is characterized by poor outcomes and mutations in the tumour suppressor p53. Our objective was to engender synthetic lethality to paclitaxel (PTX), the frontline treatment for endometrial cancer, in tumours with mutant p53 and enhance the therapeutic efficacy using polymeric nanoparticles (NPs). First, we identified the optimal NP formulation through comprehensive analyses of release profiles and cellular-uptake and cell viability studies. Not only were PTX-loaded NPs superior to PTX in solution, but the combination of PTX-loaded NPs with the antiangiogenic molecular inhibitor BIBF 1120 (BIBF) promoted synthetic lethality specifically in cells with the loss-of-function (LOF) p53 mutation. In a xenograft model of endometrial cancer, this combinatorial therapy resulted in a marked inhibition of tumour progression and extended survival. Together, our data provide compelling evidence for future studies of BIBF- and PTX-loaded NPs as a therapeutic opportunity for LOF p53 cancers.

Understanding the thermodynamics and kinetics of phosphate adsorption is crucial in the design of packed-bed columns for phosphate removal, particularly in clinical applications such as dialysis, in which limited contact time is available. Studies have shown that the kinetics of phosphate adsorption on metal oxides is a two-step process: rapid initial adsorption that occurs in a few minutes to a few hours is followed by slower adsorption that may last for days. In this work, the thermodynamics and kinetics of phosphate adsorption on lanthanum oxide impregnated on activated carbon (La-AC) are investigated to explain its phosphate adsorption behavior. The phosphate adsorption mechanism was investigated by studying (1) the effects of temperature on phosphate adsorption capacity, (2) the dynamics and enthalpy of adsorption using flow microcalorimetry (FMC), (3) the kinetics of phosphate adsorption, and (4) the relationship between the amount of phosphate adsorbed and the amount of hydroxide released. Phosphate adsorption on La-AC followed the Langmuir isotherm. FMC results indicate that phosphate adsorbed through several mechanisms, principally through the formation of monodentate and bidentate inner-sphere surface complexes. A pseudo-second-order kinetic model is typically used to model phosphate adsorption; however, it was found that a kinetic model developed based on the assumption of two parallel surface adsorption reactions fits the measured adsorption kinetics significantly better than the pseudo-second order kinetic model that is almost universally used to describe phosphate adsorption kinetics.

Objectives: Though many ovarian cancer patients respond to primary treatment, a significant portion will not respond and are considered ‘platinum resistant/refractory.’ Such patients experience a significantly worse prognosis relative to those who are ‘platinum sensitive.’ Pre-treatment prediction of platinum sensitivity has eluded scientists, which forces clinicians to undergo a ‘trial and error’ method of treatment by which resistant patients are forced to try and fail a futile line of therapy. Patient-derived organoid (PDO) cultures represent a potentially powerful functional model which preserves tumor heterogeneity (Hill et al, 2018). Here, we describe the successful culture of 4 PDOs from patients with advanced stage ovarian carcinoma and their correlation with clinical treatment outcome. Methods: Fresh tissue was obtained from the patients’ debulking surgery. It was digested, and a cell pellet was created. The pellet was washed several times, re-suspended in Matrigel, plated, and grown at 37oC over several days. PDOs were treated with standard doses of carboplatin (1 µM) and paclitaxel (10 nM). The percent of viable cells at 72 hours was measured using the CellTiter-Glo 3D platform (Promega, Madison, WI). The patients’ clinical charts were then reviewed 6 months after the completion of primary chemotherapy to determine clinical platinum sensitivity as defined by clinical parameters. Results: PDOs were successfully obtained from each of the four patients with advanced stage ovarian carcinoma. Three patients had primary disease, and 1 patient was being treated for a recurrence. The patient with recurrent disease received chemotherapy immediately prior to her debulking surgery, and the rest underwent primary debulking surgery with chemotherapy to follow. Three patients had high grade serous histology, and 1 patient had low grade serous histology. Three patients ultimately had platinum resistant disease, and their PDOs all demonstrated >50% cell viability in response to carboplatin and paclitaxel with 2 resistant PDOs demonstrating a remarkable 90% and 95% cell viability. The PDO from the patient with platinum sensitive disease by clinical criteria demonstrated 49% cell viability in response to treatment (Table 1). Download : Download high-res image (123KB) Download : Download full-size image Conclusions: We describe the successful creation of PDOs from fresh tissue obtained from debulking surgery in advanced stage ovarian cancer patients. If we use ≥50% cell viability as our initial, preliminary sensitivity threshold, results from the PDO drug sensitivity assay correlate with clinical platinum sensitivity in this pilot feasibility. We propose to confirm the predictability of drug sensitivity thresholds in future, larger studies using PDOs. Currently, there is no standardized drug sensitivity assay or response parameters defined as predictable for PDOs. Such work will be integral in the formation of a functional model of drug sensitivity, which can be used in the clinic to predict platinum sensitivity without requiring a patient to try and fail futile treatments.

Optical coherence lifetimes and decoherence processes in erbium-doped lithium niobate ( Er 3 + :LiNbO3) crystalline powders are investigated for materials that underwent different mechanical and thermal treatments. Several complimentary methods are used to assess the coherence lifetimes for these highly scattering media. Direct intensity or heterodyne detection of two-pulse photon echo techniques was employed for samples with longer coherence lifetimes and larger signal strengths, while time-delayed optical free induction decays were found to work well for shorter coherence lifetimes and weaker signal strengths. Spectral hole burning techniques were also used to characterize samples with very rapid dephasing processes. The results on powders are compared to the properties of a bulk crystal, with observed differences explained by the random orientation of the particles in the powders combined with new decoherence mechanisms introduced by the powder fabrication. Modeling of the coherence decay shows that paramagnetic materials such as Er 3 + :LiNbO3 that have highly anisotropic interactions with an applied magnetic field can still exhibit long coherence lifetimes and relatively simple decay shapes even for a powder of randomly oriented particles. We find that coherence properties degrade rapidly from mechanical treatment when grinding powders from bulk samples, leading to the appearance of amorphous-like behavior and a broadening of up to three orders of magnitude for the homogeneous linewidth even when low-energy grinding methods are employed. Annealing at high temperatures can improve the properties in some samples, with homogeneous linewidths reduced to less than 10 kHz, approaching the bulk crystal linewidth of 3 kHz under the same experimental conditions.

Objective Gynecologic Oncology Group (GOG) 177 demonstrated that addition of paclitaxel to a backbone of adriamycin/cisplatin improves overall survival (OS) and progression-free survival (PFS) for patients with advanced or recurrent endometrial cancer. Using patient specimens from GOG-177, our objective was to identify potential mechanisms underlying the improved clinical response to taxanes. Stathmin (STMN1) is a recognized poor prognostic marker in endometrial cancer that functions as a microtubule depolymerizing protein, allowing cells to transit rapidly through mitosis. Therefore, we hypothesized that one possible mechanism underlying the beneficial effects of paclitaxel could be to counter the impact of stathmin. Methods We analyzed the expression of stathmin by immunohistochemistry (IHC) in 69 specimens from patients enrolled on GOG-177. We also determined the correlation between stathmin mRNA expression and clinical outcomes in The Cancer Genome Atlas (TCGA) dataset for endometrial cancer. Results We first established that stathmin expression was significantly associated with shorter PFS and OS for all analyzed cases in both GOG-177 and TCGA. However, subgroup analysis from GOG-177 revealed that high stathmin correlated with poor PFS and OS particularly in patients who received adriamycin/cisplatin only. In contrast, there was no statistically significant association between stathmin expression and OS or PFS in patients treated with paclitaxel/adriamycin/cisplatin. Conclusions Our findings demonstrate that high stathmin expression is a poor prognostic marker in endometrial cancer. Paclitaxel may help to negate the impact of stathmin overexpression when treating high risk endometrial cancer cases.

The disruption of estrogen signaling is widely associated with the development of breast, endometrial and ovarian cancers. As a multifunctional mediator of carcinogenesis, metadherin (MTDH)/astrocyte elevated gene-1 (AEG-1) overexpression has been associated with numerous types of cancer, with reported roles in tumor initiation, proliferation, invasion, metastasis and chemoresistance. At the molecular level, MTDH has been shown to interact with proteins that drive tumorigenesis, including nuclear factor-κB (NF-κB), promyelocytic leukaemia zinc finger (PLZF), BRCA2- and CDKN1A (p21Cip1/Waf-1/mda-6)-interacting protein α (BCCIPα) and staphylococcal nuclease and tudor domain containing 1 (SND1). Through the analysis of the Cancer Genome Atlas (TCGA) datasets for estrogen receptor (ER)-positive endometrial and breast cancers, we found that over 25% of all gene expression correlated with MTDH. Using Affymetrix microarrays, we characterized the differences in gene expression between estrogen-treated parental and MTDH-deficient endometrial and breast cancer cells. We also explored a possible interaction between MTDH and ER by immunoprecipitation, and found that MTDH and ER associated in both breast and endometrial cancer cells in response to estrogen. Reciprocal co-immunoprecipitation analysis demonstrated that acute estrogen stimulation promoted the interaction of MTDH with ER in the nucleus. These data, to the best of our knowledge, provide the first evidence that MTDH and ERα interact in the nucleus with estrogen treatment to regulate gene expression.

Die Therapie der Lichturtikaria gilt als schwierig. In einigen Fallen wurde uber das gute Ansprechen auf den Anti-IgE-Antikorper Omalizumab (Xolair®), der bereits zur Therapie der chronischen spontanen Urtikaria zugelassen ist, berichtet. Wir stellen hier eine 50-jahrige Patientin vor, die seit 5 Jahren uber das lokale Auftreten von juckenden und brennenden Hautrotungen und Schwellungen unter Besonnung klagte, wobei gelegentlich auch Schockfragmente auftraten. Orale Antihistaminika in 3‑ bis 4‑facher Dosierung und die topische Anwendung eines Sunblockers hatten sich unter Langzeitfeldbedingungen nur als teilweise wirksam erwiesen. In der Lichttestung fanden sich urtikarielle Sofortreaktionen auf niedrige Dosen UVB und UVA. Drei Wochen nach s. c.-Gabe von 300 mg Omalizumab fand sich die minimale Urtikaria-auslosende Dosis (MUD) von UVB um das mindestens 20-Fache (von

Hybrid 2.5-D field solvers have been successfully applied to the analysis of high-speed printed circuit boards (PCB) and electronic packages. To enable electromagnetic interference and electromagnetic compatibility analysis, the solver needs to calculate the electromagnetic radiation in the near-field and far-field regions of the structure efficiently and accurately. We present two enhancements to the radiation calculations. The first is to use fast algorithms to accelerate the near-field and far-field calculations and the second is to correct the trace radiations by including the effect of the dielectric substrate. We demonstrate the accuracy and efficiency of these enhancements with three simulation examples.

Objective Platinum compounds have been widely used as a primary treatment for many types of cancer. However, resistance is the major cause of therapeutic failure for patients with metastatic or recurrent disease, thus highlighting the need to identify novel factors driving resistance to Platinum compounds. Metadherin (MTDH, also known as AEG-1 and LYRIC), located in a frequently amplified region of chromosome 8, has been consistently associated with resistance to chemotherapeutic agents, though the precise mechanisms remain incompletely defined. Methods The mRNA of FANCD2 and FANCI was pulled down by RNA-binding protein immunoprecipitation. Pristimerin-loaded nanoparticles were prepared using the nanoprecipitation method. Immunocompromised mice bearing patient-derived xenograft tumors were treated with pristimerin-loaded nanoparticles, cisplatin and a combination of the two. Results MTDH, through its recently discovered role as an RNA binding protein, regulates expression of FANCD2 and FANCI, two components of the Fanconi anemia complementation group (FA) that play critical roles in interstrand crosslink damage induced by platinum compounds. Pristimerin, a quinonemethide triterpenoid extract from members of the Celastraceae family used to treat inflammation in traditional Chinese medicine, significantly decreased MTDH, FANCD2 and FANCI levels in cancer cells, thereby restoring sensitivity to platinum-based chemotherapy. Using a patient-derived xenograft model of endometrial cancer, we discovered that treatment with pristimerin in a novel nanoparticle formulation markedly inhibited tumor growth when combined with cisplatin. Conclusions MTDH is involved in post-transcriptional regulation of FANCD2 and FANCI. Pristimerin can increase sensitivity to platinum-based agents in tumors with MTDH overexpression by inhibiting the FA pathway.

The utility of comprehensive surgical staging in patients with low risk disease has been questioned. Thus, a reliable means of determining risk would be quite useful. The aim of our study was to create the best performing prediction model to classify endometrioid endometrial cancer (EEC) patients into low or high risk using a combination of molecular and clinical-pathological variables. We then validated these models with publicly available datasets. Analyses between low and high risk EEC were performed using clinical and pathological data, gene and miRNA expression data, gene copy number variation and somatic mutation data. Variables were selected to be included in the prediction model of risk using cross-validation analysis, prediction models were then constructed using these variables. Model performance was assessed by area under the curve (AUC). Prediction models were validated using appropriate datasets in The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases. A prediction model with only clinical variables performed at 88%. Integrating clinical and molecular data improved prediction performance up to 97%. The best prediction models included clinical, miRNA expression and/or somatic mutation data, and stratified pre-operative risk in EEC patients. Integrating molecular and clinical data improved the performance of prediction models to over 95%, resulting in potentially useful clinical tests.

Nearly one-third of patients with high-grade serous ovarian cancer (HGSC) do not respond to initial treatment with platinum-based therapy. Genomic and clinical characterization of these patients may lead to potential alternative therapies. Here, the objective is to classify non-responders into subsets using clinical and molecular features. Using patients from The Cancer Genome Atlas (TCGA) dataset with platinum-resistant or platinum-refractory HGSC, we performed a genome-wide unsupervised cluster analysis that integrated clinical data, gene copy number variations, gene somatic mutations, and DNA promoter methylation. Pathway enrichment analysis was performed for each cluster to identify the targetable processes. Following the unsupervised cluster analysis, three distinct clusters of non-responders emerged. Cluster 1 had overrepresentation of the stage IV disease and suboptimal debulking, under-expression of miRNAs and mRNAs, hypomethylated DNA, “loss of function” TP53 mutations, and the overexpression of genes in the PDGFR pathway. Cluster 2 had low miRNA expression, generalized hypermethylation, MUC17 mutations, and significant activation of the HIF-1 signaling pathway. Cluster 3 had more optimally cytoreduced stage III patients, overexpression of miRNAs, mixed methylation patterns, and “gain of function” TP53 mutations. However, the survival for all clusters was similar. Integration of genomic and clinical data from patients that do not respond to chemotherapy has identified different subgroups or clusters. Pathway analysis further identified the potential alternative therapeutic targets for each cluster.

Objectives: Endometrial cancer incidence and mortality are rising in the US. Disease recurrence has been shown to have a significant impact on mortality. However, to date, there are no accurate and validated prediction models that would discriminate which individual patients are likely to recur. Reliably predicting recurrence would be of benefit for treatment decisions following surgery. We present an integrated model constructed with comprehensive clinical, pathological and molecular features designed to discriminate risk of recurrence for patients with endometrioid endometrial adenocarcinoma. Subjects and methods: A cohort of endometrioid endometrial cancer patients treated at our institution was assembled. Clinical characteristics were extracted from patient charts. Primary tumors from these patients were obtained and total tissue RNA extracted for RNA sequencing. A prediction model was designed containing both clinical characteristics and molecular profiling of the tumors. The same analysis was carried out with data derived from The Cancer Genome Atlas for replication and external validation. Results: Prediction models derived from our institutional data predicted recurrence with high accuracy as evidenced by areas under the curve approaching 1. Similar trends were observed in the analysis of TCGA data. Further, a scoring system for risk of recurrence was devised that showed specificities as high as 81% and negative predictive value as high as 90%. Lastly, we identify specific molecular characteristics of patient tumors that may contribute to the process of disease recurrence. Conclusion: By constructing a comprehensive model, we are able to reliably predict recurrence in endometrioid endometrial cancer. We devised a clinically useful scoring system and thresholds to discriminate risk of recurrence. Finally, the data presented here open a window to understanding the mechanisms of recurrence in endometrial cancer.

To: 1) develop/adapt and validate an instrument to measure patient safety attitudes and opinions of community-based spinal manipulative therapy (SMT) providers; 2) implement the instrument; and 3) compare results among healthcare professions.A review of the literature and content validation were used for the survey development. Community-based chiropractors and physiotherapists in 4 Canadian provinces were invited.The Agency for Healthcare Research and Quality's (AHRQ) Medical Office Survey on Patient Safety Culture was the preferred instrument. The survey was modified and validated, measuring 14 patient safety dimensions. 276 SMT providers volunteered to respond to the survey. Generally, SMT providers had similar or better patient safety dimension scores compared to the AHRQ 2016 medical offices database.We developed the first instrument measuring patient safety attitudes and opinions of community-based SMT providers. This instrument provides understanding of SMT providers' opinions and attitudes on patient safety and identifies potential areas for improvement.1) Élaborer/adapter et valider un instrument servant à évaluer les attitudes à l’égard de la sécurité du patient et les opinions des praticiens effectuant des manipulations vertébrales (MV); 2) adopter cet instrument; et 3) comparer les résultats obtenus entre les professionnels de la santé.Pour élaborer le sondage, on a revu la littérature, on a validé le contenu et on a invité des chiropraticiens et des physiothérapeutes de quatre provinces canadiennes à participer.On a élaboré le premier instrument servant à évaluer les attitudes à l’égard de la sécurité et les opinions des praticiens effectuant des MV dans une collectivité. Cet instrument permet de comprendre les opinions et les attitudes à l’égard de la sécurité du patient des professionnels effectuant des MV et de cerner les aspects qui pourraient être améliorés.

Simple Summary Microorganisms are found in all human tissues. Some of them are responsible for cancer formation. In our study we found gene expression from bacteria, archaea, and viruses in the upper female genital tract and this expression was associated with ovarian and endometrial cancer. We also found that the expression from these organisms may be involved in regulatory mechanisms of infection and cancer formation. Some of the processes associated with these organisms may affect cancer heterogeneity and be potential targets for cancer therapy. Abstract Bacteria, archaea, and viruses are associated with numerous human cancers. To date, microbiome variations in transcription have not been evaluated relative to upper female genital tract cancer risk. Our aim was to assess differences in bacterial, archaea, and viral transcript (BAVT) expression between different gynecological cancers and normal fallopian tubes. In this case-control study we performed RNA sequencing on 12 normal tubes, 112 serous ovarian cancers (HGSC) and 62 endometrioid endometrial cancers (EEC). We used the centrifuge algorithm to classify resultant transcripts into four indexes: bacterial, archaea, viral, and human genomes. We then compared BAVT expression from normal samples, HGSC and EEC. T-test was used for univariate comparisons (correcting for multiple comparison) and lasso for multivariate modelling. For validation we performed DNA sequencing of normal tubes in comparison to HGSC and EEC BAVTs in the TCGA database. Pathway analyses were carried out to evaluate the function of significant BAVTs. Our results show that BAVT expression levels vary between different gynecological cancers. Finally, we mapped some of these BAVTs to the human genome. Numerous map locations were close to regulatory genes and long non-coding RNAs based on the pathway enrichment analysis. BAVTs may affect gynecological cancer risk and may be part of potential targets for cancer therapy.

Introduction: GOG-086P was one of the first attempts to combine molecular inhibitors such as bevacizumab or temsirolimus with chemotherapy in patients with advanced endometrial cancer. An exploratory analysis of this study indicates that bevacizumab combined with chemotherapy is superior to temsirolimus plus chemotherapy in patients with TP53 mutated tumors as determined by next generation sequencing. The functional status of p53 in a tumor can be inferred by sequence analysis and/or by immunohistochemistry (IHC). In this next study of the GOG-086P cohort, we sought to determine whether p53 IHC alone or integrated with TP53 sequencing was similarly predictive of outcome and whether IHC was predictive of the sequence analysis. Methods: A total of 349 patients were enrolled on GOG-086P. Two hundred and forty-three patients have TP53 sequence data, and 213 have p53 protein expression data measured by IHC. Tumor histology and sequence data were correlated with p53 IHC categorizing each case as p53 negative or null, wild type (WT) or over-expressed (OE), and these variables were further correlated with progression-free survival (PFS) and overall survival (OS) in the chemotherapy + bevacizumab arms versus the chemotherapy + temsirolimus arm. Results: The majority of patients (118) had tumors with WT p53 protein expression by IHC and non-mutated or WT TP53 sequence, with expected concordance between IHC and sequence analysis. However, there were five discordant cases with OE p53 protein in the absence of a TP53 mutation. Fourteen patients had null IHC protein expression and a concordant TP53 sequence mutation expected to result in the total loss of protein. Four cases that were p53 null by IHC had a discordant WT TP53 sequence. Seventeen patients had tumors with a missense TP53 mutation but had WT p53 protein expression by IHC, reflecting a potential discordance between sequencing and IHC versus a missense mutation that did not result in loss of protein function. Six of these 17 discordant cases may be explained by co-mutations in TP53 and POLE or microsatellite instability where p53 proteins, though mutated, function more like WT. Reassuringly and as expected, high concordance was found in the 55 cases that had OE protein by IHC and a missense mutation in TP53. On integrated analysis, a TP53 mutation with p53 protein OE was associated with significantly lower hazard ratio for PFS (HR=0.41; 95% CI 0.22-0.83) and OS (HR=0.28; 95% CI 0.14-0.59), i.e., longer PFS and OS, for patients receiving bevacizumab + chemotherapy relative to temsirolimus + chemotherapy. Conclusions: Mutations in TP53, which are common in patients with advanced endometrial cancer, represent a developing platform upon which to design personalized treatment regimens. We demonstrate that IHC is often but not always predictive of the TP53 sequence. However, most cases with over-expressed p53 protein by IHC had a mutation in TP53, and bevacizumab + chemotherapy prolonged PFS and OS in such patients. Citation Format: Kristina W. Thiel, Eric J. Devor, Virginia Filiaci, Douglas A. Levine, Fanny Dao, Narciso Olvera, David Mutch, Carol Aghajanian, Kimberly K. Leslie. Correlation of immunohistochemistry with TP53 sequence and clinical outcomes in GOG-086P [abstract]. In: Proceedings of the AACR Virtual Special Conference: Endometrial Cancer: New Biology Driving Research and Treatment; 2020 Nov 9-10. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(3_Suppl):Abstract nr PR006.

Patient-derived organoids (PDOs) are a promising model for personalized cancer treatment. Accumulating evidence indicates that PDOs can predict clinical outcomes in patients. A handful studies in several cancer types have established that PDOs can recapitulate both histological and genomic features of the lesion from which they were derived. Additionally, PDOs can grow with high efficiency in a short time, enabling prediction of responsiveness to chemotherapy and development of more targeted therapies for patients. Our long-term goal is to create a PDO biorepository comprising all major histopathological subtypes of endometrial cancer, perform drug screening on these PDOs, and identify novel single agent and combinatorial regimens to advance into clinical trials. Herein we describe progress to date on generation of endometrial PDOs and drug screening to identify potential therapeutic options. First, we created PDO cultures from seven patients with different histological subtypes of endometrial cancer, including early stage/grade endometrioid adenocarcinoma and high-grade serous carcinoma. Next, we exposed each PDO to a panel of 23 chemotherapeutic agents and targeted drugs. Surprisingly, despite a divergent response to standard chemotherapy (platinum and taxane compounds), all PDOs showed high responsiveness to the combination of a histone deacetylase (HDAC) inhibitor and a proteasome inhibitor. Specifically, we achieved nearly complete cell killing within 72 hrs of exposure of organoid cultures to HDAC and proteasome inhibitors, with concentrations in the low nanomolar range. Similar results were achieved with different proteasome inhibitors (bortezomib and ixazomib) and HDAC inhibitors (romidepsin and belinostat). At present, these classes of agents have primarily been studied in multiple myeloma, though multiple clinical trials of combined proteasome and HDAC inhibitor therapy are active in advanced solid tumors and lymphomas. HDAC inhibitors maintain the acetylation of histones and thereby increase the expression of genes associated with apoptosis and cell cycle arrest. Proteasome inhibitors prevent proteasomal degradation, leading to upregulation of proapoptotic protein expression, cell cycle arrest, and ER stress, downregulation of angiogenesis and the pro-inflammatory protein NF-κB, and ROS generation. Mechanistically, HDAC inhibitors and proteasome inhibitors promote dual proteasome and aggresome blockage and induce apoptosis due to the accumulation of misfolded proteins. Our findings of profound tumor cell death in multiple different PDOs serve as the foundation for a future clinical trial using the HDAC inhibitor ixazomib and proteasome inhibitor romidepsin in advanced and recurrent endometrial cancers as well as high-grade serous ovarian cancer, which shares histologic and molecular features. Concomitantly, we are investigating the in vitro and in vivo effects of the combination on the induction of apoptosis and endometrial tumor regression. Citation Format: Jianling Bi, Kristina W. Thiel, Eric J. Devor, Andreea M. Newtson, Yuping Zhang, Kimberly K. Leslie. Combined histone deacetylase and proteasome inhibition in patient-derived endometrial cancer organoid models promotes massive cell death [abstract]. In: Proceedings of the AACR Virtual Special Conference: Endometrial Cancer: New Biology Driving Research and Treatment; 2020 Nov 9-10. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(3_Suppl):Abstract nr PO039.

Progesterone prevents development of endometrial cancers through its receptors A (PRA) and B (PRB) although the molecular mechanisms have yet to be fully characterized. In this study, we performed a global analysis of gene regulation by progesterone using human endometrial cancer cells that express heterologous progesterone receptors. Functional replicate Affymetrix microarrays encompassing 54,000 transcripts were performed for two independent experiments. Data suggest that progesterone exerts the growth-limiting effects in endometrium via down-regulation of several growth factors important in angiogenesis and cell proliferation. In particular, progesterone down-regulates the vascular endothelial growth factor (VEGF), platelet derived growth factor (PDGF), and signal transducer and activator of transcription 3 (STAT3) genes. Down-regulation of these pro-growth pathways required the co-expression of both PR isoforms A and B, whereas expression of either isoform alone resulted in unchanged or even elevated levels of PDGF and STAT3 transcripts. Our study identifies these pathways as central to the growth limiting effects of progesterone in endometrial cancer and suggests that these factors constitute important targets for future therapeutic interventions. We also provide a comprehensive catalogue of the transcriptional effects of PR isoforms in endometrial cancer for future reference. Citation Format: Kimberly K. Leslie, Stephanie M. Leiva, Donghai Dai, Yuping Zhang, Kristina W. Thiel. A comprehensive analysis of the transcriptional effects of progesterone receptor isoforms A and B identifies VEGF, PDGF and STAT3 as therapeutic targets [abstract]. In: Proceedings of the AACR Virtual Special Conference: Endometrial Cancer: New Biology Driving Research and Treatment; 2020 Nov 9-10. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(3_Suppl):Abstract nr PO005.

Angiogenesis plays a crucial role in tumor development and metastasis, and many cancer cells upregulate vascular endothelial growth factor-A (VEGF-A) expression to promote angiogenesis. Several clinical trials of anti-angiogenic agents have been conducted in advanced and recurrent endometrial cancer, but only bevacizumab and cediranib have demonstrated activity as single agents. Bevacizumab is a monoclonal antibody against VEGF-A, whereas cediranib is a small molecule angiokinase inhibitor. However, subsequent studies of bevacizumab+chemotherapy failed to improve outcomes compared to chemotherapy alone. We recently demonstrated that chemotherapy plus nintedanib, another angiokinase inhibitor, promotes catastrophic cell death in a xenograft model of endometrial cancer (Ebeid, et al., Nat Nanotech 2019). This effect was specific to cells with loss of function, i.e., null mutations in TP53. Here we compared the efficacy of cediranib vs. bevacizumab in p53-null gynecologic cancer models and determined the mechanism for differential sensitivity. We first performed a phosphoproteomic array of 875 phosphoproteins to define the signaling changes related to bevacizumab vs. cediranib in two p53-null cell gynecologic cancer cell lines. Several signaling events were similar between bevacizumab and cediranib, including phosphorylation of IGF1R, Abl, p70S6K and proteins in the ERK/MAPK and Wnt signaling pathways. We next tested the impact of the anti- angiogenic agents on cell viability using cancer cell lines and over 20 patient-derived organoid cultures of endometrial or ovarian cancer. Neither bevacizumab nor cediranib alone had a notable effect on cell viability, even at 1-10 µM concentrations. By contrast, cediranib but not bevacizumab promoted marked cell death when combined with chemotherapy. This effect was most pronounced in p53-null models. Cell cycle analysis demonstrated an accumulation in mitosis after treatment with cediranib+chemotherapy, consistent with abrogation of the G2/M checkpoint and subsequent mitotic catastrophe. Molecular analysis of key controllers of the G2/M cell cycle checkpoint confirmed its abrogation. Unexpectedly, the endometrial cancer cell lines were unresponsive to stimulation with VEGF-A, the target of bevacizumab. This was confirmed by the lack of ERK phosphorylation, a downstream signaling event expected after VEGF-A treatment, and was potentially caused low expression of VEGFR1 and 2. Based on these data, we conclude that an anti-angiogenic tyrosine kinase inhibitor such as cediranib has the potential to be superior to bevacizumab in combination with chemotherapy. We hypothesize that the mechanism relates to cediranib inhibition of the tumor vasculature as well as its ability to optimize the impact of chemotherapeutic agents during the cell cycle. Future clinical studies should test this hypothesis by studying the combination of standard chemotherapy with cediranib in advanced and recurrent endometrial cancer. Citation Format: Jianling Bi, Andreea M. Newtson, Eric J. Devor, Yuping Zhang, Kristina W. Thiel, Kimberly K. Leslie. Anti-angiogenic tyrosine kinase inhibitor cediranib is superior to bevacizumab in endometrial cancer due to differential effects on cell cycle regulation [abstract]. In: Proceedings of the AACR Virtual Special Conference: Endometrial Cancer: New Biology Driving Research and Treatment; 2020 Nov 9-10. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(3_Suppl):Abstract nr PO046.

Introduction: NRG Study GY011 is a surgical window trial of medroxy-progesterone acetate (MPA) +/- entinostat (E), a histone deacetylase inhibitor hypothesized to upregulate PR levels through epigenetic modifications. The principal goal of this trial was to determine the impact of treatment on the level as well as the activity of progesterone receptors (PR) in women with endometrial cancer. However, a reliable marker for PR activity was needed, and the most promising choice was Ki-67. To be approved for inclusion in the study as an integrated biomarker, Ki-67 had to first undergo rigorous evaluation by the participating pathologists with oversight by the NIH Biomarker Review Committee. Methods: Fifty patients were enrolled on GY011, and specimen collection was planned by biopsy prior to receiving study drugs and by tissue sample at the time of the hysterectomy. There were 42 pre-drug administration specimens with tumor for analysis and 1 without and 43 hysterectomy specimens with tumor. Ki-67 immunohistochemistry performed on pre and post drug specimens was evaluated by three experienced reviewers (MS, KC and AR) using a qualitative approach, blinded to treatment arm. Pre and post-drug samples for each individual patient were compared and scored as increased, decreased, or unchanged. The Fleiss kappa statistic was chosen as the measure of concordance as it allows for more than two reviewers and more than two categories. Percent agreement and standard weighted kappa statistics were calculated for individual pairs of reviewers. Results: There was very good concordance between reviewers using the qualitative comparison of post to pre-drug Ki-67 staining with Fleiss’ Kappa of 0.91 and weighted Kappa’s ranging between 0.85 and 0.95. Reviewers 1 and 2 had the highest level of concordance. Reviewer agreement ranged from 0.92 to 0.98. The reviewer-specific (marginal) score distributions were similar. Each reviewer scored about 80% (79.5%-82.5%) of pairs as decreasing, with the remainder split similarly between no change (7.5%-10%) and increasing (10%-12.8%). Conclusion: Based upon the high concordance between reviewers, the NIH Biomarker Review Committee approved Ki-67 as an integrated biomarker for GY011, setting the stage for its use as an approved biomarker of therapeutic effectiveness in future endometrial cancer trials. Citation Format: Megan I. Samuelson, Virginia Filiaci, Kelley Carrick, Anand Rajan KD, William H. Rodgers, Kristina W. Thiel, Linda R. Duska, Kimberly K. Leslie. Approval for the proliferative marker Ki-67 as an integrated biomarker for endometrial cancer in NRG study GY011 [abstract]. In: Proceedings of the AACR Virtual Special Conference: Endometrial Cancer: New Biology Driving Research and Treatment; 2020 Nov 9-10. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(3_Suppl):Abstract nr PO020.

The appropriate models to use for studies of cancer development, progression, drug response and resistance to therapy remain hotly contested. Leading models include cell lines, genetically-modified mice, patient-derived organoids and patient-derived xenografts (PDX). Progress in studies of endometrial cancer is hampered in part by the lack of extensively characterized research models. Our group recently performed next-generation sequencing on the five most commonly-studied endometrial cancer cell lines, Ishikawa H, ECC1, Hec50co, KLE and RL95-2. Our data, which includes histology, mutation screening, MLH1 promoter methylation, copy number variation and microsatellite instability (MSI), conclude that none of the five models cleanly fit any extant post-hoc classification scheme, including the four clusters defined by TCGA: POLE ultra-mutated (cluster 1), MSI hypermutated (cluster 2), copy-number low (endometrioid, cluster 3), and copy-number high (serous-like, cluster 4). We hypothesized that cell lines may not fully represent the true biology of endometrial cancer. Therefore, we have now established PDX models of endometrial cancer in hopes of creating a more representative set of research tools. We generated PDX models by implanting fresh endometrial tumor tissue (hysterectomy specimens) subcutaneously into athymic mice. Of 21 implanted tumors, 8 successfully grafted (take rate=38%). After the initial growth in mice (passage 1 or P1), tumors could be excised, cryopreserved and stored for extended periods in liquid nitrogen. Cryopreserved tumors retain the original phenotype of the P1 engrafted PDX tumor, maintain the ability to proliferate and can be utilized for subsequent larger-scale xenograft studies. Histology is preserved for 5-7 passages, including the architectural pattern of abnormal glands, sheets and occasional papillary configurations. Additionally, some PDX models could be cultured in 2D. We next undertook an extensive morphologic and molecular characterization of three PDX models, denoted EC-PDX1, 2 and 3. EC-PDX1 is serous by histology but contains a POLE mutation (P286R) along with mutations in MSH2, ARID1A and TP63, suggestive of a cluster 1 POLE ultramutated phenotype. Expression of estrogen and progesterone receptors (ER and PR) were low or absent. EC-PDX1 was sensitive to cisplatin in vivo. EC-PDX2 is a high-grade endometrioid model devoid of ER, PR and p53 by immunohistochemistry. Mutational analysis identified a frame shift mutation in exon 8 of p53, which results in a premature stop codon and supports the null staining. These data suggest that EC-PDX2 is most similar to copy-number high cluster 4. EC-PDX3 is low-grade endometrioid with robust expression of ER and PR and wild-type levels of p53. This model harbors activating mutations in PI3KCA (E545K) and KRAS (G13D) and a stop gained mutation in ARID1A. Based on these features, we bin EC-PDX3 as a copy-number low cluster 3. We conclude that PDX models may be superior to cancer cell lines for some studies of the biology and therapeutic response of endometrial cancer. Citation Format: Yuping Zhang, Eric J. Devor, Donghai Dai, Jesus Gonzalez-Bosquet, Kristina W. Thiel, Kimberly K. Leslie. Characterization of patient-derived xenograft models of endometrial cancer [abstract]. In: Proceedings of the AACR Virtual Special Conference: Endometrial Cancer: New Biology Driving Research and Treatment; 2020 Nov 9-10. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(3_Suppl):Abstract nr PR009.

Advanced stage serous carcinoma of the uterus typically portends a poor prognosis. There is an urgent need to augment clinical trial data with different methods of treatment evaluation before a patient is made to try and potentially fail a therapy. Patient derived organoids (PDOs) may represent such an opportunity. We describe the successful culture and drug sensitivity testing of two PDOs from patients with advanced stage serous carcinoma of the uterus who received neoadjuvant chemotherapy. Clinical response to treatment was compared with the organoid response to the same regimens. Drug sensitivity data are reported as percentage of residual cell viability 48 hours after one standard treatment dose, in which 100% viability indicates no response and 0% viability reflects complete cell death. We hypothesized that significant cell killing, i.e. a low cell viability in our assay, would be required to achieve clinical drug sensitivity in patients. Both patients were clinically platinum resistant. Patient 1 had a large burden of disease at the time of presentation, including skeletal and liver metastasis. Though she initially partially responded to triplet therapy of paclitaxel, carboplatin and bevacizumab, she ultimately had persistent unresectable disease after 6 cycles of therapy. The percent residual, post-drug cell viability for PDO-1 was 45% in response to paclitaxel, carboplatin and bevacizumab combinatorial treatment, 35% in response to paclitaxel and carboplatin, and 100% in response to single agent bevacizumab. Patient 2 received paclitaxel, carboplatin and trastuzumab because her tumor was positive for HER2neu. Though she also had an initial response to treatment, she experienced a recurrent malignant pleural effusion 3 months after completing platinum-containing triplet (trastuzumab was continued as a maintenance therapy). The percent residual cell viability for PDO-2 was 51% in response to the combination of paclitaxel and carboplatin. Previous investigators have identified PDOs as a potentially powerful functional model for treatment response. We confirm that it is feasible to establish organoids and to rapidly test agents for cell sensitivity even prior to the first post-surgical chemotherapeutic treatment cycle. For both patients, the PDO models were only moderately sensitive to the agents used for therapy, and the lack of long-term benefit achieved for these patients may have been suggested a priori by the PDO models. However, further studies are necessary to establish a threshold percent value of post-treatment cell viability that is predictive of clinical patient response. We hypothesize that such a threshold appears to be significantly below 50% from these two initial cases. Citation Format: Andreea M. Newtson, Jianling Bi, Yuping Zhang, Eric Devor, Kristina Thiel, Kimberly K. Leslie. Development of patient derived organoid cultures of two advanced stage serous carcinomas of the uterus [abstract]. In: Proceedings of the AACR Virtual Special Conference: Endometrial Cancer: New Biology Driving Research and Treatment; 2020 Nov 9-10. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(3_Suppl):Abstract nr PO025.