Pantarhei Bioscience (Netherlands)

Background: The co-treatment of androgen deprivation therapy (ADT) for advanced prostate cancer (PCa) with the fetal estrogen estetrol (E4) may further inhibit endocrine PCa tumor stimulators. We previously reported the suppression of follicle-stimulating hormone (FSH), total and free testosterone, and prostate-specific antigen by ADT+E4. Here, we provide more detailed data on FSH suppression by E4 and present new findings on the effect of ADT+E4 on insulin-like growth factor-1 (IGF-1). Methods: A Phase II, double-blind, randomized, placebo-controlled study (the PCombi study) was conducted in advanced PCa patients treated with ADT. The study assessed the effect of E4 co-treatment with LHRH agonist ADT on tumor stimulators, including FSH and IGF-1. Patients starting ADT were randomized 2:1 to receive either 40 mg E4 (n = 41) or placebo (n = 21) for 24 weeks. Non-parametric analyses were performed on the per-protocol population (PP) and individual changes were visualized. Results: The PP included 57 patients (37 ADT+E4; 20 ADT+placebo). ADT+E4 almost completely suppressed FSH in all patients (98% versus 37%; p < 0.0001). IGF-1 levels decreased by 41% with ADT+E4 versus an increase of 10% with ADT+placebo (p < 0.0001). Conclusions: The almost complete suppression of the tumor stimulator FSH using ADT plus E4 observed in all individual patients in this study, along with the augmented suppression of IGF-1 versus an increase by ADT only, may be clinically relevant and suggest the enhanced anti-cancer treatment efficacy of E4 in addition to the previously reported additional suppression of total and free T and PSA.

Abstract Introduction Effective drug development relies on representative pre-clinical models to discover and characterize novel cancer therapies. Recapitulating the human specific tumor micro-environment (TME) is essential for IO studies or TME-targeting agents and detection of human-specific (off-)target effects to better predict therapeutic response and clinical outcomes. The recently developed patient-derived organoid (PDO) technology, provides access to representative organotypic cultures, established from patient cancer stem cells. These cultures can be further improved by including additional cell types with strong clinical implications, especially cancer-associated fibroblasts (CAFs) and immune cells. Both CAFs and immune cells from various lineages modulate tumor growth and metastasis, drug resistance and immune-mediated cancer cell killing in the TME and have become popular targets for therapy in the recent decades. Here, we present our improved pre-clinical platform for drug testing, consisting of organotypic mini-tumors combined with high-content imaging screening (HCI) to discriminate different cell types and assess morphological, cytostatic and cytotoxic effects of drugs simultaneously. Methods 3D mini-tumor co-cultures were established of allogenic CAFs and PDOs from pancreatic, colon and breast cancer patients. The mini-tumors were expanded in a TME stimulating medium cocktail that drives myofibroblast expansion and differentiation through PDGFRβ and TGFβ signaling. Subsequently, drug responses to various standard of care (SoC) treatments were determined. Additionally, allogenic PBMCs were included in mini-tumor cultures to assess mini-tumor infiltration and killing. Results Mini-tumor co-cultures were successfully established and characterized in different stimulating cocktails. We found mini-tumor sensitivity to SoC treatment to be altered compared to PDO mono-cultures or co-cultures without TME stimulating factors. Moreover, we observed changes in PBMC infiltration and cancer cell killing in mini-tumors grown in TME stimulating conditions. Conclusion and discussion Taken together, our newly developed pre-clinical mini-tumor HCI platform provides the unique opportunity to screen and test drugs targeting human cancer cells, CAFs or immune cells in vitro in a pathology and TME-resembling context. This will ultimately result in effective target validation and mode of action analysis, timeline and cost reductions, and most importantly, a more efficient entry into the clinic. Citation Format: Marten Hornsveld, Guusje Odenkirchen, Cyrelle van Dort, Jara Garcia Mateos, Saskia de Man, Gera Goverse, Bram Herpers, Mariusz Madej, Leo Price, Marrit Putker. Mini-tumors: Pre-clinical HCI assays to screen anti-cancer drugs in the context of organoids, cancer-associated fibroblasts and immune cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 579.

Abstract This study describes the expression of an intracellular variant of the Zona Pellucida glycoprotein 3 (ZP3) that is predominantly expressed in cancer. ZP3 is an important constituent of the Zona Pellucida (ZP), the extracellular layer surrounding oocytes. It is highly expressed in maturing oocytes, but not in other adult human tissues. As ZP3 protein has also been detected in cancer, it may serve as an interesting diagnostic and therapeutic target. Here, we extend the cancer-related ZP3 expression data using immunohistochemistry (IHC) of tumor biopsies, interrogating publicly available RNA-sequencing (RNA-seq) data of cancer cell lines (CCLs) and tumor and normal tissues, as well as computational analysis and real-time quantitative PCR (qPCR) of CCLs. IHC data for several cancer types shows abundant ZP3 protein expression, which is confined to the cytoplasm, contradicting the extracellular ZP3 localization in oocytes. An alternative ZP3 mRNA variant, which we term ‘ZP3-Cancer’, is annotated in the NCBI and Ensembl databases, and lacks the genetic information encoding the N-terminal signal peptide that governs extracellular secretion. Analysis of publicly available RNA-seq data of 1339 CCLs indicates that ZP3-Cancer is the dominant variant as compared to ZP3-Oocyte, which was validated by independent computational analysis. Expression of ZP3-Cancer in CCLs was confirmed by qPCR. Publicly available RNAseq data of tumor and normal tissues confirms strongly enhanced expression of ZP3-Cancer in cancer cells. In addition, ZP3-Cancer expression is upregulated in advanced stages and shows a relationship with patient survival in a number of cancer types. The folliculogenesis specific transcription factor FIGLA, which activates transcription of ZP3-Oocyte in maturing oocytes, appears absent in cancer as inferred from publicly available transcriptome data, indicating alternative ZP3-Cancer transcriptional activation mechanisms in this disease. The cancer restricted expression of this ZP3 transcript variant renders it an attractive tumor specific antigen for the development of a therapeutic cancer vaccine, particularly using mRNA technology. Citation Format: Iman J. Schultz, Yvette Zimmerman, Cathy B. Moelans, Marcin Chrusciel, Jan Krijgh, Paul J. van Diest, Ilpo T. Huhtaniemi, Herjan J. Coelingh Bennink. An intracellular variant of zona pellucida glycoprotein 3 is expressed in cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3937.

Objectives The aim of the study was to evaluate the efficacy of different dosages of estetrol (E4) combined with one of two progestins in suppressing the pituitary–ovarian axis and ovulation in healthy premenopausal women.Methods This was an open, parallel, phase II, dose-finding, pilot study performed in healthy women aged 18 to 35 years with a documented ovulatory cycle before treatment. For three consecutive cycles in a 24/4-day regimen, participants received 5 mg or 10 mg E4/3 mg drospirenone (DRSP); 5 mg, 10 mg or 20 mg E4/150 μg levonorgestrel; or 20 μg ethinylestradiol (EE)/3 mg DRSP as comparator. Pituitary–ovarian axis activity and the occurrence of ovulation were evaluated by monitoring follicular size, serum levels of follicle-stimulating hormone, luteinising hormone, estradiol and progesterone during treatment cycles 1 and 3. Endometrial thickness was evaluated throughout the trial, and the return of ovulation was evaluated after the last intake of medication.Results A total of 109 women were included in the trial. No ovulation occurred in any treatment group. Ovarian activity inhibition seemed proportional to the E4 dosage: the highest suppression was observed in the 20 mg E4 group and was very similar to that observed with EE/DRSP. Endometrial thickness was suppressed to the same extent in all groups. Post-treatment ovulation occurred in all participants between 17 and 21 days after the last active treatment. The study combinations were well tolerated and safe.Conclusions Combined with a progestin, E4 adequately suppresses ovarian activity, particularly when given at a dosage above 10 mg/day.Chinese 摘要目的 这项研究的目的是评估不同剂量的雌四醇联合两种孕激素中的其中一种对垂体-卵巢轴以及健康的绝经前妇女的排卵方面的抑制疗效。方法 这是一个在18到35岁的健康女性中进行的开放的、平行的，关于II期药物剂量探索的初步研究，这些女性均有治疗前的排卵周期记录。连续三个周期的24/4-天方案,参与者接受5毫克或10毫克的雌四醇/3毫克屈螺酮;5毫克,10毫克或20毫克的雌四醇/150 μg左炔诺孕酮;或用20 μg炔雌醇/3毫克屈螺酮作比较。在第1和第3个治疗周期，通过监测卵泡的大小,血清促卵泡激素、黄体生成素、雌二醇和孕激素的水平对垂体-卵巢轴的活动和排卵进行评估。对子宫内膜厚度的评估贯穿于整个试验过程中，对排卵抑制的评估在用完药物之后。结果 总共有109名妇女参与试验。任何一个治疗组都没有出现排卵。对卵巢活动的抑制似乎与雌四醇的剂量成正比：最严重的抑制出现在20毫克的雌四醇组，与在炔雌醇/屈螺酮组观察到的情况非常相似。所有组的子宫内膜厚度的抑制程度是相同的。在最后一次积极治疗后的第17到21天之间，所有参与者均出现了治疗后的排卵。所有的试验组合均有良好的耐受性和安全性。结论 雌四醇联合一种雌激素能够充分的抑制卵巢活动，尤其是当雌四醇的剂量大于10毫克/天的时候。关键词雌四醇；雌激素；口服避孕药；排卵抑制；孕激素

Objectives To determine whether human sex hormone binding globulin (SHBG) binds estetrol (E4), and to assess whether E4 stimulates the production of SHBG by human hepatocytes.Methods Competitive ligand binding assays have been used to assess the relative binding affinity of E4 to human SHBG using either [3H]5α-dihydrotestosterone or [3H]estradiol as labeled ligands. The effect of E4 on the production of SHBG has been assessed by a fluoroimmunometric assay in wild-type human HepG2 cells and in human Hep89 cells that over-express the human estrogen receptor (ER) α, and compared to the effect of ethinylestradiol, estradiol and estriol.Results There was no detectable binding of E4 to the human SHBG steroid-binding sites. By contrast, testosterone and estradiol were bound with high affinity and the synthetic estrogen ethinylestradiol was found to bind SHBG with low affinity. Estetrol does not stimulate ERα-mediated increases in SHBG production by HepG2 or Hep89 cells, in contrast to ethinylestradiol, estradiol and estriol.Conclusions These data indicate that SHBG has no influence on the plasma distribution of E4 or its availability to target tissues. In addition, it is shown that E4 has no effect on SHBG production by human hepatocytes.

In this Appendix, data are reported obtained by in vitro studies, performed with the human fetal steroid estetrol (E4). The studies have been executed by a commercial laboratory in Taiwan (MDS). Es...