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MET, the cell-surface receptor for the hepatocyte growth factor/scatter factor, which is widely overexpressed in various solid cancer types, is an attractive target for the development of antibody-based therapeutics. BYON3521 is a novel site-specifically conjugated duocarmycin-based antibody–drug conjugate (ADC), comprising a humanized cysteine-engineered IgG1 monoclonal antibody with low pmol/L binding affinity towards both human and cynomolgus MET. In vitro studies showed that BYON3521 internalizes efficiently upon MET binding and induces both target- and bystander-mediated cell killing. BYON3521 showed good potency and full efficacy in MET-amplified and high MET–expressing cancer cell lines; in moderate and low MET–expressing cancer cell lines good potencies and partial efficacy were observed. In mouse xenograft models, BYON3521 showed significant antitumor activity upon single-dose administration in multiple non-MET–amplified tumor types with low, moderate, and high MET expression, including complete tumor remissions in models with moderate MET expression. In the repeat-dose Good Laboratory Practice (GLP) safety assessment in cynomolgus monkeys, BYON3521 was well tolerated and based on the observed toxicities and their reversibility, the highest non-severely toxic dose was set at 15 mg/kg. A human pharmacokinetics (PK) model was derived from the PK data from the cynomolgus safety assessments, and the minimal efficacious dose in humans is estimated to be in the range of 3 to 4 mg/kg. In all, our nonclinical data suggests that BYON3521 is a safe ADC with potential for clinical benefit in patients. A first-in-human dose-escalation study is currently ongoing to determine the maximum tolerated dose and recommended dose for expansion (NCT05323045).

MET, the cell-surface receptor for the hepatocyte growth factor/scatter factor, which is widely overexpressed in various solid cancer types, is an attractive target for the development of antibody-based therapeutics. BYON3521 is a novel site-specifically conjugated duocarmycin-based antibody–drug conjugate (ADC), comprising a humanized cysteine-engineered IgG1 monoclonal antibody with low pmol/L binding affinity towards both human and cynomolgus MET. In vitro studies showed that BYON3521 internalizes efficiently upon MET binding and induces both target- and bystander-mediated cell killing. BYON3521 showed good potency and full efficacy in MET-amplified and high MET–expressing cancer cell lines; in moderate and low MET–expressing cancer cell lines good potencies and partial efficacy were observed. In mouse xenograft models, BYON3521 showed significant antitumor activity upon single-dose administration in multiple non-MET–amplified tumor types with low, moderate, and high MET expression, including complete tumor remissions in models with moderate MET expression. In the repeat-dose Good Laboratory Practice (GLP) safety assessment in cynomolgus monkeys, BYON3521 was well tolerated and based on the observed toxicities and their reversibility, the highest non-severely toxic dose was set at 15 mg/kg. A human pharmacokinetics (PK) model was derived from the PK data from the cynomolgus safety assessments, and the minimal efficacious dose in humans is estimated to be in the range of 3 to 4 mg/kg. In all, our nonclinical data suggests that BYON3521 is a safe ADC with potential for clinical benefit in patients. A first-in-human dose-escalation study is currently ongoing to determine the maximum tolerated dose and recommended dose for expansion (NCT05323045).

Supplementary tables and figures

Table S1. Summary of status of tumors used in cell line and patient-derived xenograft models. Table S2. SYD985 and T-DM1 IC50, 95% confidence interval and % efficacy values from the in vitro cytotoxicity assays as depicted in Figure 1B. Table S3. Pharmacokinetic parameters for ADC (SYD985 and T-DM1) in BT-474-tumor-bearing mice after a single i.v. bolus injection of ADC. Figure S1. In vitro cytotoxicity. Figure S2. A-G HER2 expression in BT-474. Figure S3. HER2 cell surface expression quantified on a panel of 8 cell lines using the DAKO Qifikit. Figure S4. In vitro cytotoxicity. Figure S5. In vitro bystander killing. Figure S6. Mean ADC (SYD985 conjugated antibody) concentrations in plasma in carboxylesterase CES1c knock-out (-/-) , CES1c heterozygeous (+/-) and CES1c wild type (+/+) mice, after single i.v. bolus injection of SYD985 at 5 mg/kg (+/- SEM, n = 3).

Carboxylesterase 1c (CES1c) is responsible for linker-drug instability and poor pharmacokinetics (PK) of several antibody–drug conjugates (ADC) in mice, but not in monkeys or humans. Preclinical development of these ADCs could be improved if the PK in mice would more closely resemble that of humans and is not affected by an enzyme that is irrelevant for humans. SYD985, a HER2-targeting ADC based on trastuzumab and linker-drug vc-seco-DUBA, is also sensitive to CES1c. In the present studies, we first focused on the interaction between CES1c and SYD985 by size- exclusion chromatography, Western blotting, and LC/MS-MS analysis, using recombinant CES1c and plasma samples. Intriguingly, CES1c activity not only results in release of the active toxin DUBA but also in formation of a covalent bond between CES1c and the linker of vc-seco-DUBA. Mass spectrometric studies enabled identification of the CES1c cleavage site on the linker-drug and the structure of the CES1c adduct. To assess the in vivo impact, CES1c−/− SCID mice were generated that showed stable PK for SYD985, comparable to that in monkeys and humans. Patient-derived xenograft (PDX) studies in these mice showed enhanced efficacy compared with PDX studies in CES1c+/+ mice and provided a more accurate prediction of clinical efficacy of SYD985, hence delivering better quality data. It seems reasonable to assume that CES1c−/− SCID mice can increase quality in ADC development much broader for all ADCs that carry linker-drugs susceptible to CES1c, without the need of chemically modifying the linker-drug to specifically increase PK in mice. Mol Cancer Ther; 17(11); 2389–98. ©2018 AACR.

Figure S1: In vitro kinetics of SYD985. Total antibody concentration during 96-hour incubation of 100 mg/mL SYD985 at 37°C in human, monkey, rat and mouse plasma (mean +/- SEM). Figure S2: Ion trap MS/MS fragmentation spectrum (HCD @30 eV) of three SYD985-CES1c cross-linked peptide structures (all precursor ions were 3+) Figure S3: A, Conjugated antibody concentrations in female BALB/c mice after a single IV bolus injection of 1 and 5 mg/kg SYD985 in combination with an intraperitoneal injection of 150 mg/kg BNPP, 15 minutes prior to SYD985 dosing (mean +/- SEM, n=3). B, Conjugated antibody concentrations in female BALB/c mice after a single IV bolus injection of 5 mg/kg SYD985 in combination with increasing subcutaneous dosages of CBDP, 60 minutes prior to SYD985 dosing (mean +/- SEM, n=3). Figure S4: Anti-tumor activity of SYD985 compared to vehicle control in the breast cancer PDX models MAXF449. Table S1: ADC pharmacokinetic parameters in CES1c transgenic mice mice after a single i.v. bolus injection of 5 mg/kg SYD985. Table S2: IC50 and efficacy values of cytotoxic activity presented in Figure 1E. Table S3: Comparison of SYD985 conjugated antibody PK parameters in MAXF1162 tumor bearing CES1c -/- SCID mice with the PK in human.

Engineering cysteines at specific sites in antibodies to create well-defined ADCs for the treatment of cancer is a promising approach to increase the therapeutic index and helps to streamline the manufacturing process. Here, we report the development of an

Carboxylesterase 1c (CES1c) is responsible for linker-drug instability and poor pharmacokinetics (PK) of several antibody–drug conjugates (ADC) in mice, but not in monkeys or humans. Preclinical development of these ADCs could be improved if the PK in mice would more closely resemble that of humans and is not affected by an enzyme that is irrelevant for humans. SYD985, a HER2-targeting ADC based on trastuzumab and linker-drug vc-seco-DUBA, is also sensitive to CES1c. In the present studies, we first focused on the interaction between CES1c and SYD985 by size- exclusion chromatography, Western blotting, and LC/MS-MS analysis, using recombinant CES1c and plasma samples. Intriguingly, CES1c activity not only results in release of the active toxin DUBA but also in formation of a covalent bond between CES1c and the linker of vc-seco-DUBA. Mass spectrometric studies enabled identification of the CES1c cleavage site on the linker-drug and the structure of the CES1c adduct. To assess the in vivo impact, CES1c−/− SCID mice were generated that showed stable PK for SYD985, comparable to that in monkeys and humans. Patient-derived xenograft (PDX) studies in these mice showed enhanced efficacy compared with PDX studies in CES1c+/+ mice and provided a more accurate prediction of clinical efficacy of SYD985, hence delivering better quality data. It seems reasonable to assume that CES1c−/− SCID mice can increase quality in ADC development much broader for all ADCs that carry linker-drugs susceptible to CES1c, without the need of chemically modifying the linker-drug to specifically increase PK in mice. Mol Cancer Ther; 17(11); 2389–98. ©2018 AACR.

The pipelines of pharmaceutical companies are filled with thousands of promising new compounds for a plethora of indications. Yet, a close review of the drugs that have recently been in clinical trials quickly reveals that only a handful of drugs under evaluation in women with endometriosis can be genuinely qualified as truly innovative and breakthrough drugs. Why is there such an industry-wide lukewarm interest in drug research and development for endometriosis/adenomyosis? Why are pharmaceutical companies so reluctant to initiate programs or invest in academic research in endometriosis/adenomyosis? It is evident that a substantial part of the novel druggable targets originate from research in academia. However, only the pharmaceutical industry has the resources and expertise to bring drugs to patients. In other words, we are fully dependent on the pharmaceutical industry to bring new therapeutics to the market. The aim of this editorial is to make scientists from academia aware of the enormous complexity of the drug development process, the drivers that propel pharmaceutical companies to initiate new programs and to prioritize their portfolios, the value of intellectual property rights, and also about the importance of scientific rigor, predictive translational models, and biomarkers. At the same time, the pharmaceutical industry must be made aware of the enormous opportunity at hand, as the current patient population with endometriosis/adenomyosis is just the tip of the iceberg. We hope that the insights presented here will enable the endometriosis/adenomyosis research community to find ways to valorize their knowledge and attract the interest of the industry.

Background The drug research and development (R&D) for endometriosis/adenomyosis has been painfully slow. Most completed clinical trials on endometriosis did not publish their results, and presumably failed. While few published trials did report how they foundered, the reasons why they failed are often completely unclear. Surprisingly, there has been no open discussion on why these trials failed. If the causes for these failed trials remain unelucidated, mistakes made in these failed trials may be repeated in the future. Since failure can be infinitely more instructive and educational than success, elucidating the causes for failed clinical trials may yield a treasure trove for future drug R&D. Given our growing understanding of the natural history of ectopic endometrium, it is also important to make an inventory of biologicals/compounds that are currently under development to see where we stand and whether they would stand a better chance of gaining regulatory approval than their predecessors. Objective and rationale We provide an overview of all compounds under clinical investigation and in development in order to assess the evolution of R&D since the last inventory, reported in 2013. We also have attempted to analyse selected failed clinical trials in the context of published translational/preclinical research and our growing understanding of the natural history of endometriotic/adenomyotic lesions, in the hope that the lessons learned will steer investigators toward the right track in future drug R&D. Search methods We searched ClinicalTrials.gov and a database containing information on drugs gathered daily by Thomson Reuters from a wide range of sources (e.g. patent offices, biomedical literature, congresses, symposia, meetings, company information, regulatory information) for all therapeutic compounds that have undergone or are under clinical trials, or in the developmental stage, and then searched PubMed and Google to determine their publication status using trial identifiers. For trials that were completed at least 2 years ago and have, or have not, published their results, a PubMed search was performed using the name of the therapeutic that has been tested and 'endometriosis' or 'adenomyosis' to identify published preclinical studies prior to the launch of the trial. For those published trials, the cited preclinical studies were also retrieved and scrutinized. Outcomes Despite repeated calls for more transparency, only a small fraction of completed trials on endometriosis has been published. A large number of 'novel' compounds under development are simply repurposed drugs, which seem to be ill-prepared to combat the fibroproliferative nature of endometriosis/adenomyosis. This sobering picture indicates an alarming innovation 'drought' in the drug R&D front, resulting in trickling drug pipelines. Some trials foundered owing to unanticipated serious side-effects, or because attempts were made to suppress a target that can be compensated for by redundant pathways, but many failed in efficacy, indicating that the translational value of the current models is seriously questionable. All existing animal models of endometriosis do not recapitulate the key features of human conditions. Wider implications The glaring innovation drought in drug R&D for endometriosis/adenomyosis should sound alarms to all stake-holders. The failed clinical trials in endometriosis also indicate that some past research had serious deficiencies. In light of the recent understanding of the natural history of ectopic endometrium, it is perhaps time to shift the research paradigm and revamp our research focus and priorities.

Item does not contain fulltext Chronic pain and subfertility are the main symptoms of concern in women with endometriosis. In order to find new therapeutic options to suppress the pain, translational animal models are indispensable. We have developed a new automated, experimental setup, with full consideration for animal wellbeing, to determine whether operant behaviour can reveal abdominal hyperalgesia in rats with surgically-induced endometriosis, in order to assess whether abdominal hyperalgesia affect behavioural parameters. Endometriosis was induced by transplantation of uterine fragments in the abdominal cavity. Control groups consisted of sham-operated rats and non-operated rats. We have developed an operant chamber (Skinnerbox) which includes a barrier. The rat can climb the barrier in order to reach the food pellet, increasing in this way the pressure to the abdomen. We show that endometriosis rats collect significantly less sugar pellets when compared with the control rats after the introduction of the barrier. In the Skinnerbox experiment, we showed that in a positive operant setting, the introduction of a barrier results in a contrast of operant behaviour of endometriosis rats and control groups, perchance as a result of abdominal discomfort/hyperalgesia due to surgically-induced endometriosis. This is a promising start for the further development of a refined animal model to monitor abdominal discomfort/hyperalgesia in rats with surgically-induced endometriosis. 8 p.

Simple Summary Soft tissue sarcoma (STS) is a heterogeneous group of hard-to-treat malignancies from mesenchymal or connective tissues. The treatment options in case the cancer relapses after surgery or is at an advanced state are limited, and in all cases the response rates are low. Targeted therapy with antibody drug conjugates may provide an alternative. To this end, molecules that are specifically (over)expressed on the surface of the tumor cells are needed. In this study, we show that 5T4/trophoblast glycoprotein, a molecule with limited expression in healthy tissues, is prominently expressed on the cell surface of many STS subtypes and can be used for such an approach. Abstract The expression of 5T4/trophoblast glycoprotein was evaluated in several histological subtypes of soft tissue sarcoma (STS) to determine whether the prevalence and level of expression of this membrane-associated glycoprotein is sufficient for use in targeted therapies. Tumor tissue microarrays containing cores from different histological subtypes of STS were stained using a standardized immunohistochemical staining method to detect 5T4; the level of staining was assessed using a semi-quantitative scoring method. No 5T4 staining was seen in the angiosarcomas and liposarcomas investigated in this study. 5T4 staining in the other STS subtypes was seen in more than 50% of cases, warranting further investigation into whether this antigen could evoke an anti-tumor immune response or can be used as target for the delivery of more potent toxins through antibody drug conjugates.

5T4 is an oncofetal antigen with low expression in healthy tissues which is frequently overexpressed in different tumor types. Despite its overexpression in various malignancies and its association with poor prognosis, there are currently no marketed drugs that target this tumor antigen. We developed a novel antibody-drug conjugate (ADC), named SYD1875, comprising a humanized, HC41-cysteine-engineered IgG1 monoclonal antibody directed against 5T4, site-specifically conjugated to a proprietary cleavable synthetic duocarmycin-based linker-drug (vc-seco-DUBA). SYD1875 showed sub-nanomolar binding affinity to human and cynomolgus 5T4, rapid internalization, and induction of both target- and bystander-mediated cell killing. SYD1875 showed superior in vitro cytotoxicity and in vivo activity compared to A1-mcMMAF -also known as PF-6263507-, a 5T4-targeting ADC that is no longer in clinical development. A single dose of SYD1875 induced strong anti-tumor activity in patient-derived xenograft models of multiple tumor types with low, moderate and high 5T4 expression. The GLP toxicity study in cynomolgus monkey showed an acceptable toxicity and PK profile. A first-in-human dose-finding trial was initiated to evaluate safety and explore efficacy (NCT04202705). Citation Format: Patrick Groothuis, Danielle Jacobs, Kim Berentsen, Monique van der Vleuten, Ruud Coumans, Ronald Elgersma, Marion Blomenrohr, Diels van den Dobbelsteen, Patrick Beusker, Ruud Ubink, Miranda van der Lee, Wim H.A. Dokter. Introduction to the preclinical profile of SYD1875, a novel site-specifically conjugated duocarmycin-based 5T4-targeting antibody-drug conjugate [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 925.

c-Met, the cell-surface receptor for HGF/SF which is widely overexpressed in various solid cancer types, is an attractive target for the development of antibody-based therapeutics. However, the apparently delicate balance between anti-tumor activity and target mediated-toxicities proved to be quite a challenge. As a result, there are currently no c-Met targeting antibodies or antibody-drug conjugates (ADCs) on the market. We developed BYON3521, a novel site-specifically conjugated, duocarmycin-based ADC, comprising a humanized, cysteine-engineered IgG1 monoclonal antibody with low pM binding affinity towards both human and cynomolgus c-Met. In vitro studies showed that BYON3521 internalizes efficiently upon c-Met binding, and induces both target- as well as bystander-mediated cell killing. BYON3521 showed good potency and full efficacy in MET-amplified and high c-Met-expressing cancer cell lines; in moderate and in low c-Met-expressing cancer cell lines partial efficacy was observed. PK/PD studies and a mouse clinical trial in patient-derived xenograft models showed significant anti-tumor activity of BYON3521 upon single dose administration in multiple tumor types. BYON3521 showed responses in non-MET-amplified tumors with low, moderate and high c-Met expression, with partial and complete tumor remission seen in several models with moderate c-Met expression. In the repeat-dose GLP safety assessment in cynomolgus monkeys BYON3521 was well tolerated. In all, BYON3521 was deemed a safe ADC with potential for clinical benefit in patients. Preparations are ongoing to start clinical Phase I in 2021. Citation Format: Patrick Groothuis, Danielle Jacobs, Inge Hermens, Desiree Damming, Ellen Mattaar, Myrthe Rouwette, Monique van der Vleuten, Aloys Sesink, Fred Dijcks, Ruud Coumans, Marion Blomenrohr, Ruud Ubink, Miranda van der Lee, Wim H.A. Dokter. BYON3521, a novel effective and safe c-Met targeting antibody-drug conjugate [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 926.

SYD985 is a HER2-targeting antibody-drug conjugate comprised of a cleavable linker and the prodrug seco-DUBA which, upon release, spontaneously rearranges to form the active toxin DUBA. DUBA covalently binds adenine in the DNA resulting in DNA damage, cell cycle perturbation and associated cytotoxicity. SYD985 induces dose-dependent cytotoxicity of human tumor cell lines and PDXs expressing HER2 on the outer cell membrane. The cytotoxicity of PARPi depends on PARP trapping, i.e. the formation of non-covalent protein-DNA adducts composed of inhibited PARP1 bound to DNA lesions of clear1 and unclear origins2,3. We investigated potential synergy in cytotoxic activity of SYD985 and the PARPi niraparib and olaparib towards HER2-expressing tumor cells in vitro and in vivo. Tumor cell lines were from breast (BT-474, AU-565, UACC-893) or ovarian (TOV-112D, NIH:OVCAR-3) origin. Cytotoxicity was established by measuring DNA content and metabolic activity of living cells. For both PARPi the highest non-cytotoxic concentration for each cell line was established. This concentration was used to measure the potential potency shift of the SYD985 concentration-response curve by comparing SYD985 IC50 values in the absence and presence of PARPi. PARPi-induced fold change in IC50 for SYD985 was established in two independent experiments (N=2, n=3). Niraparib, dosed at a non-cytotoxic concentration, induced a 1.5 to 136-fold potentiation of SYD985-induced tumor cell killing. In BT-474 cells, niraparib co-treatment unveils a biphasic concentration-response curve to SYD985. This is in line with the fact that BT-474 cells express high levels of HER2 and are known to respond to naked trastuzumab at concentrations slightly above those of SYD985. Olaparib, dosed at a non-cytotoxic concentration, induced a 0.4 to 7.5-fold potentiation of SYD985-induced tumor cell killing. In a breast cancer PDX mouse clinical trial in immunodeficient mice that lack carboxylesterase 1c (Ces1c) we studied the combination of SYD985 (1 mg/kg, IV, SD) and niraparib or olaparib (50 mg/kg Q1DX21, IP). In order to detect potential additive and/or synergistic effects of SYD985 and PARPi, a suboptimal dose of SYD985 (1 mg/kg) was selected. PARPi doses were chosen known to inhibit growth of tumors with a defective DNA repair mechanism4. Data clearly show that the combination of SYD985 and PARPi are more effective in tumor volume reduction than either monotherapy alone. In conclusion, niraparib and olaparib act in synergy with SYD985 in killing human HER2-expressing tumor cells in vitro, both in high HER2 (BT-474, AU-565, UACC-893) and low HER2 (NIH:OVCAR-3; TOV-112D) expressing cells. In vivo such synergistic activity appears to translate in improved tumor growth inhibition of various breast cancer PDX models. 1Zimmermann M, Nature 559, 2018; 2Pommier Y, Sci Transl Med 8, 2016; 3Lord C, Science 355, 2017; 4Henneman L, PNAS 112, 2015 Citation Format: Wim H. Dokter, Fred A. Dijcks, Patrick G. Groothuis, Ellen Mattaar, Eline Loosveld, Daniëlle Jacobs, Monique van der Vleuten, Jan H. Schellens, Miranda van der Lee. SYD985 synergy with PARP inhibitors (PARPi) in human tumor cell lines and patient-derived xenografts (PDXs) [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4043.

SYD985 is a novel anti-HER2 antibody-drug conjugate (ADC) in development for breast cancer. The ADC consists of three parts: i) the monoclonal antibody trastuzumab, ii) a linker that can be cleaved by tumor-resident proteases at the dipeptide valine-citrulline (vc) motif, and iii) the prodrug seco-DUocarmycin-hydroxyBenzamide-Azaindole. The average ‘drug to antibody ratio’ (DAR) of this ADC, also named trastuzumab vc-seco-DUBA, is ∼2.8. After antibody-mediated binding of SYD985 to its molecular target HER2 on the surface of tumor cells, the ADC is internalized. Subsequently, the active duocarmycin is released and binds to DNA in the minor groove, followed by alkylation of the DNA and killing of the tumor cells. In vitro and in vivo experiments were performed to (directly) compare various pharmacodynamic and pharmacokinetic properties of SYD985 with those of the most progressed (marketing-approved) HER2-targeting ADC, i.e. ado-trastuzumab emtansine (T-DM1). T-DM1 is currently indicated for second line treatment of breast cancer patients overexpressing HER2 tumor tissue. In in vitro experiments, SYD985 shows potencies similar to T-DM1 in cell lines expressing HER2 at high levels (IHC HER2 3+), whereas SYD985 is significantly more potent compared to T-DM1 in cell lines expressing low HER2 levels (IHC HER2 2+ or 1+). In line with these results, SYD985 has superior efficacy compared to T-DM1 in xenograft models in which patient-derived breast cancer tumor tissue with low HER2 levels (IHC HER2 2+ or 1+/FISH-) have been used. Pharmacokinetic experiments (in vitro and in vivo) have revealed that whereas in mice SYD985 has limited stability, in cynomolgus monkey SYD985 is very stable. Excellent stability in vitro is also observed in human plasma. Moreover, after i.v. administration of SYD985 in the cynomolgus monkey the amount of free toxin (DUBA) detected in the monkey plasma is many-fold lower than levels reported for the toxin (DM1) released from T-DM1. Resuming, the preclinical data of SYD985 support clinical studies to investigate whether SYD985 has benefit in cancer patients with moderate or even low HER2 levels of the tumor tissue. Citation Format: Gijs Verheijden, Patrick Beusker, Ruud Ubink, Miranda van der Lee, Patrick Groothuis, Peter Goedings, David Egging, Marco Timmers, Wim Dokter. Preclinical data of SYD985 support the clinical investigation of this novel anti-HER2 antibody-drug conjugate in breast cancer patients with low levels of HER2 expression [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P4-15-14.

SYD985 is a HER2-targeting antibody–drug conjugate (ADC) based on trastuzumab and vc-seco-DUBA, a cleavable linker-duocarmycin payload. To evaluate the therapeutic potential of this new ADC, mechanistic in vitro studies and in vivo patient-derived xenograft (PDX) studies were conducted to compare SYD985 head-to-head with T-DM1 (Kadcyla), another trastuzumab-based ADC. SYD985 and T-DM1 had similar binding affinities to HER2 and showed similar internalization. In vitro cytotoxicity assays showed similar potencies and efficacies in HER2 3+ cell lines, but in cell lines with low HER2 expression, SYD985 was 3- to 50-fold more potent than T-DM1. In contrast with T-DM1, SYD985 efficiently induced bystander killing in vitro in HER2-negative (HER2 0) cells mixed with HER2 3+, 2+, or 1+ cell lines. At pH conditions relevant for tumors, cathepsin-B cleavage studies showed efficient release of the active toxin by SYD985 but not by T-DM1. These in vitro data suggest that SYD985 might be a more potent ADC in HER2-expressing tumors in vivo, especially in low HER2-expressing and/or in heterogeneous tumors. In line with this, in vivo antitumor studies in breast cancer PDX models showed that SYD985 is very active in HER2 3+, 2+, and 1+ models, whereas T-DM1 only showed significant antitumor activity in HER2 3+ breast cancer PDX models. These properties of SYD985 may enable expansion of the target population to patients who have low HER2-expressing breast cancer, a patient population with still unmet high medical need. Mol Cancer Ther; 14(3); 692–703. ©2015 AACR.

Objective To identify metabolites that are associated with and predict the presence of endometriosis. Design Metabolomics study using state-of-the-art mass spectrometry approaches. Setting University hospital and universities. Patient(s) Twenty-five women with laparoscopically confirmed endometriosis (cases) and 19 women with laparoscopically documented absence of endometriosis (controls). None of the women included in this study had received oral contraception or GnRH agonists for a minimum of 1 month before blood collection. Intervention(s) Plasma collection. Main Outcome Measure(s) Metabolite profiles were generated and interrogated using multiple mass spectrometry methods, that is, high performance liquid chromatography coupled with negative mode electrospray ionization tandem mass spectrometry, UPLC-MS/MS, and ultra performance liquid chromatography-electroSpray ionization-quadrupole time-of-flight (UPLC-ESI-Q-TOF). Metabolite groups investigated included phospholipids, glycerophospholipids, ether-phospholipids, cholesterol-esters, triacylglycerol, sphingolipids, free fatty acids, steroids, eicosanoids, and acylcarnitines. Result(s) A panel of acylcarnitines predicted the presence of endometriosis with 88.9% specificity and 81.5% sensitivity in human plasma, with a positive predictive value of 75%. However, due to data limitations the outcome of the receiver operating characteristic curve analysis was not significant. Conclusion(s) A diagnostic model based on acylcarnitines has the potential to predict the presence and stage of endometriosis.

BACKGROUND: In this study, we characterized the fibromuscular (FM) tissue, typical of deeply infiltrating endometriosis, investigated which cells are responsible for the FM reaction and evaluated whether transforming growth factorb (TGF-b) signaling is involved in this process. METHODS: FM differentiation and TGF-b signaling were assessed in deeply infiltrating endometriosis lesions (n 5 20) and a nude mouse model of endometriosis 1, 2, 3 and 4 weeks posttransplantation. The FM reaction was evaluated by immunohistochemistry using different markers of FM and smooth muscle cell differentiation (vimentin, desmin, alpha-smooth muscle actin, smooth muscle myosin heavy chain). TGF-b signaling was assessed by immunostaining for its receptors and phosphorylated Smad. RESULTS: Deeply infiltrating endometriosis lesions contain myofibroblast-like cells that express multiple markers of FM differentiation. Expression of TGF-b receptors and phospho-Smad was more pronounced in the endometrial component of the lesions than in the FM component. In the nude mouse model, alpha-smooth muscle actin expression was observed in murine fibroblasts surrounding the lesion, but not in human endometrial stroma. CONCLUSIONS: FM differentiation in deeply infiltrating endometriosis is the result of a reaction of the local environment to the presence of ectopic endometrium. It shares characteristics with pathological wound healing, but cannot be explained by TGF-b signaling alone.

It is widely known that angiogenesis plays a key role in endometriotic lesion formation and development. Antiangiogenic treatments aimed at inhibiting new vessel formation have proven efficient in experimental models. However, as antiangiogenic strategies do not target pre-existing pericyte-protected vessels, they require chronic administration and are likely to be beneficial for early-stage disease only or to prevent recurrence after surgery. Moreover, they may have detrimental effects on reproductive function. Vascular-disrupting agents (VDAs) have emerged as a promising new tool for the treatment of tumors. VDAs target established blood vessels, resulting in tumor ischemia and necrosis. These agents may therefore be more efficient against advanced disease. Two major types of VDAs are being developed for cancer: ligand-directed VDAs using antibodies, peptides and growth factors to deliver toxic effectors to tumor endothelium; and small-molecule VDAs exploiting physiological differences between tumor and normal endothelium to induce acute vascular shutdown. The ongoing evolution in genomics and proteomics is revolutionizing the discovery of novel endothelial markers. Several studies suggest that the vasculature of endometriotic lesions may have particular pathophysiological properties, which could be exploited for the development of selective VDAs. The aim of this review is to explore the merits and limitations of vascular therapy for the treatment of endometriosis.

BACKGROUND: The general concept that haemoglobin is only a carrier protein for oxygen and carbon dioxide is challenged since recent studies have shown haemoglobin expression in non-erythroid cells and the protection of haemoglobin against oxidative and nitrosative stress. Using microarrays, we previously showed expression of haemoglobins alpha, beta, delta and gamma and the haeme metabolizing enzyme, haeme oxygenase (HO)-1 in human endometrium. METHODS: Using real-time quantitative PCR, haemoglobin alpha, beta, delta and gamma, and HO-1 mRNA levels were assessed throughout the menstrual cycle (n = 30 women). Haemoglobin and HO-1 protein levels in the human endometrium were assessed with immunohistochemistry. For steroid responsiveness, menstrual and late proliferative-phase endometrial explants were cultured for 24 h in the presence of vehicle (0.1% ethanol), estradiol (17 beta-E-2, 1 nM), progestin (Org 2058, 1 nM) or 17 beta-E-2+Org 2058 (1 nM each). RESULTS: All haemoglobins and the HO-1 were expressed in normal human endometrium. Haemoglobin mRNA and protein expression did not vary significantly during the menstrual cycle. Explant culture with Org 2058 or 17 beta-E-2+Org 2058 increased haemoglobin gamma mRNA expression (P < 0.05). HO-1 mRNA levels, and not protein levels, were significantly higher during the menstrual (M)-phase of the cycle (P < 0.05), and were down-regulated by Org 2058 in M-phase explants and by 17 beta-E-2+Org 2058 in LP-phase explants, versus control (P < 0.05). CONCLUSIONS: The haemoglobin-HO-1 system may be required to ensure adequate regulation of the bioavailability of haeme, iron and oxygen in human endometrium.

BACKGROUND: Estradiol (E(2)) is an important promoter of the growth of both eutopic and ectopic endometrium. The findings with regard to the expression and activity of steroidogenic enzymes in endometrium of controls, in endometrium of endometriosis patients and in endometriotic lesions are not consistent. METHODS: In this study, we have looked at the mRNA expression and protein levels of a range of steroidogenic enzymes [aromatase, 17beta-hydroxysteroid dehydrogenases (17beta-HSD) type 1, 2 and 4, estrogen sulfotransferase (EST) and steroid sulfatase (STS)] in eutopic and ectopic endometrium of patients (n = 14) with deep-infiltrative endometriosis as well as in disease-free endometrium (n = 48) using real-time PCR and immunocytochemistry. In addition, we evaluated their menstrual cycle-related expression patterns, and investigated their steroid responsiveness in explant cultures. RESULTS: Aromatase and 17beta-HSD type 1 mRNA levels were extremely low in normal human endometrium, while mRNAs for types 2 and 4 17beta-HSD, EST and STS were readily detectable. Only 17beta-HSD type 2 and EST genes showed sensitivity to progesterone in normal endometrium. Types 1 and 2 17beta-HSD and STS protein was detected in normal endometrium using new polyclonal antibodies. CONCLUSIONS: In endometriosis lesions, the balance is tilted in favor of enzymes producing E(2). This is due to a suppression of types 2 and 4 17beta-HSD, and an increased expression of aromatase and type 1 17beta-HSD in ectopic endometrium.

Using cDNA microarray analysis, we identified SGK1 (serum- and glucocorticoid-regulated kinase 1) as a gene aberrantly expressed in midsecretory endometrium of women with unexplained infertility. SGK1 is a serine/threonine kinase involved primarily in epithelial ion transport and cell survival responses. Real-time quantitative PCR analysis of a larger, independent sample set timed to coincide with the period of uterine receptivity confirmed increased expression of SGK1 transcripts in infertile women compared with fertile controls. We further demonstrate that SGK1 expression is regulated by progesterone in human endometrium in vivo as well as in explant cultures. During the midsecretory phase of the cycle, SGK1 mRNA and protein were predominantly but not exclusively expressed in the luminal epithelium, and expression in this cellular compartment was higher in infertile women. In the stromal compartment, SGK1 expression was largely confined to decidualizing cells adjacent to the luminal epithelium. In primary culture, SGK1 was induced and phosphorylated upon decidualization of endometrial stromal cells in response to 8-bromo-cAMP and progestin treatment. Moreover, overexpression of SGK1 in decidualizing cells enhanced phosphorylation and cytoplasmic translocation of the forkhead transcription factor FOXO1 and inhibited the expression of PRL, a major decidual marker gene. Conversely, knockdown of endogenous SGK1 by small interfering RNA increased nuclear FOXO1 levels and enhanced PRL expression. The observation that SGK1 targets FOXO1 in differentiating human endometrium, together with its distinct temporal and spatial expression pattern and increased expression in infertile patients, suggest a major role for this kinase in early pregnancy events.

To date, research into the biological processes and molecular mechanisms associated with endometrial receptivity and embryo implantation has been a focus of attention, whereas the complex events that occur in the human endometrium during the menstrual and proliferative phase under the influence of estrogen have received little attention. The objective of this review is to provide an update of our current understanding of the actions of estrogen on both human and rodent endometrium, with special emphasis on the regulation of uterine growth and cell proliferation, and the value of global gene expression analysis, in increasing understanding of these processes.

Tumor hypoxia can trigger the induction of angiogenesis. High microvessel density (MVD) as well as hypoxia-inducible factor-1alpha (HIF-1alpha) have been related to recurrent disease and tumor aggressiveness, respectively. In this study, MVD and hypoxic status were investigated in primary and recurrent endometrial carcinomas. A total of 65 primary tumors of patients with recurrent endometrial carcinoma (n = 40), and without recurrent endometrial carcinoma (n = 25) were studied. Immunohistochemical analysis was performed on paraffin-embedded tumor tissue. MVD was determined by quantitative analysis of CD31/FVIII positive vessels. Tumor hypoxia was estimated by evaluating the expression of the hypoxia-regulated gene HIF-1alphaand its target gene carbonic anhydrase IX (CA-IX). An additional 23 recurrent tumors were available for determination of MVD and HIF-1alpha expression. Effects of hypoxia on tumor protein p53 (TP53) expression were evaluated in the endometrial cancer cell lines (ECC-1), Ishikawa (derived from adenocarcinomas), and AN3CA (derived from a lymph node metastasis). MVD, CA-IX, and HIF-1alpha expression were not significantly different in primary tumors of patients with recurrence compared to the control tumors. The MVD was significantly lower, and HIF-1alpha expression was significantly higher in recurrent tumors when compared with their primary tumors (paired t test, P < 0.05). HIF-1alpha expression correlated well with TP53 expression levels in primary tumors, but not in recurrences. TP53 protein levels were highest in AN3CA cells. Hypoxic conditions induced TP53 protein in ECC-1 and Ishikawa, but not AN3CA cells. We conclude that MVD, CA-IX, and HIF-1alpha expression are not independent prognostic markers for recurrent endometrial carcinoma. The low MVD, increased HIF-1alpha protein levels, dissociation of hypoxia, and TP53 protein induction in the metastatic tumor cells (AN3CA) support a role for hypoxia in the development of recurrent endometrial carcinoma.

Objective. To study alterations within the p53 pathway in relation to the development of recurrent stage I endometrioid endometrial carcinoma. Methods. Paraffin-embedded tumor tissue of both primary and recurrent tumors from 44 patients with and 44 without recurrence was used for immunohistochemical analysis of TP53, hMdm2, P21 Waf1/Cip1 and M30. DNA was extracted, and mutation analysis of p53 (exon 5–8, 11) was performed by direct sequencing. Results. TP53 overexpression was significantly associated with recurrent disease: Odds Ratio 3.8 (95% CI: 1.5–9.8). Overexpression of TP53 was associated with lower staining indices (SI:0–9) of both hMdm2 and P21 in tumors of patients with recurrence, compared to controls: 2.0 ± 0.4 vs. 4.0 ± 0.8 and 1.9 ± 0.8 vs. 3.6 ± 0.8, respectively. Eight p53 missense mutations were identified in six patients with recurrence and two controls. One nonsense mutation was found in a patient with recurrence and one deletion in a control patient. Only a minority of TP53 overexpression cases could be explained by the presence of these p53 mutations. Conclusion. TP53 overexpression was significantly predictive for recurrent endometrial carcinoma, and mostly not correlated with p53 mutations. Concomitant low hMdm2 and P21 Waf1/Cip1 expression in tumors with overexpressed TP53 suggests a dysfunctional TP53-P21 Waf1/Cip1 pathway.

Infertility is an increasing problem all over the world, and it has been estimated that 10–15% of couples in fertile age have fertility problems. Likewise induced unsafe abortion is a serious threat to women’s health. Despite advances made in assisted reproduction techniques, little progress has been made in increasing the success rate during fertility treatment. This document describes a wide range of projects carried out to increase the understanding in the field of embryo implantation research. The ‘Fruitful’ research network was created to encourage collaborations within the consortium and to describe our different research potentials to granting agencies or private sponsors.

The aim of this study was to evaluate the ex vivo oestrogen responsiveness of human proliferative phase endometrium using short-term explant cultures. The effects of oestrogen (17beta-E2) on proliferation and the expression of oestrogen-responsive genes known to be involved in regulating endometrial function were evaluated. Three distinct response patterns could be distinguished: (1) the menstrual (M) phase pattern (cycle days 2-5), which is characterised by a complete lack in the proliferative response to 17beta-E2, while an increased expression of AR (2.6-fold, P0.01), PR (2.7-fold, P0.01) and COX-2 (3.5-fold, P0.01) at the mRNA level was observed and a similar upregulation was also found for AR, PR and COX-2 at the protein level; (2) the early proliferative (EP) phase pattern (cycle days 6-10) with 17beta-E2 enhanced proliferation in the stroma (1.7-fold, P0.05), whereas the expression of AR, PR and COX-2 were not affected at the mRNA and protein levels and ER-alpha mRNA and protein levels were significantly reduced by 17beta-E2; (3) the late proliferative (LP) phase pattern (cycle days 11-14), which is characterised by a moderate stimulation of proliferation (1.4-fold, P0.05) and PR mRNA expression (1.7-fold, P0.01) by 17beta-E2. In conclusion, three distinct response patterns to 17beta-E2 could be identified with respect to proliferation and the expression of known oestrogen-responsive genes in human proliferative phase endometrium explant cultures.

Peritoneal endometriosis is the result of ectopic implantation and growth of endometrium tissue that has been regurgitated into the abdominal cavity during menstruation. We have previously shown that menstrual effluent induces epithelial to mesenchymal transitions (EMT) in mesothelial cells, which results in cell retraction and exposure of submesothelial extracellular matrix. Since endometrial tissue preferentially adheres to the extracellular matrix, adhesion of endometrial tissue to the peritoneum is facilitated. The EMT were shown to be associated with differential expression and phosphorylation of mesothelial proteins. Using radiolabeling and proteomics we detected changes in protein expression and phosphorylation that occur in mesothelial cells during the EMT process. The identity of 73 proteins, which were obtained from 324 analyzed spots, was confirmed. The expression of 35 proteins involved in organization of the cytoskeleton, signal transduction, regulation of the redox state, and production of ATP, was altered during the EMT process. Four of the identified proteins were differentially phosphorylated: annexin-1, an actin-binding protein and a substrate for receptor tyrosine kinases; tropomyosin-alpha, a regulator of actin filament stability and cell shape; elongation factor 1 delta; ATP synthase beta-chain. In conclusion, factors from menstrual effluent induce specific changes in the expression and phosphorylation status of structural, regulatory and metabolic proteins relevant to the complex process of EMT in mesothelial cells.

It is known that angiogenesis is of pivotal importance for the development of endometriosis. However, in the treatment of endometriosis patients, prevention of endometriosis lesion development only will not be sufficient as a therapy. Treatment options, aimed at interfering with established lesions, have to be developed. In this study we evaluated whether inhibition of angiogenesis by angiostatic therapy is also effective in antagonizing the sustentation of endometriosis. We evaluated the effect of the angiostatic compounds antihuman vascular endothelial growth factor, TNP-470, endostatin, and anginex on the growth of established endometriosis lesions in the nude mouse model. We show that human endometrium in the proliferative endometrium is highly angiogenic and that vascular endothelial growth factor-A is the most important angiogenesis promotory factor. The angiostatic compounds significantly decreased microvessel densities and the number of established endometriosis lesions. In the remaining lesions, the number of pericyte-protected vessels is not different in control and treated mice; however, the number of unprotected vessels was significantly reduced in the groups treated with the angiostatic agents. Our data demonstrate that inhibitors of angiogenesis effectively interfere with the maintenance and growth of endometriosis by inhibiting angiogenesis. This suggests that the use of angiostatic agents may be promising as a therapy for endometriosis.

Our understanding of the mechanisms of the actions of oestrogens and progestins have evolved from the simple concept of nuclear receptor-mediated regulation of transcription to a highly sophisticated, finely tuned interplay between various coregulators, other signaling cascades and transcription factors. The net result of these complex regulatory mechanisms is a steroid-, cell-, or tissue-specific action of oestrogens and progestins, their antagonists or selective modulators of their receptors. In this review, we have attempted to shed some light on the regulation of the actions of oestrogens and progestins on the human endometrium.

Previous in-vitro studies have shown that the endometrium preferentially adheres to the extracellular matrix (ECM) of the amnion and peritoneum. This interaction probably involves adhesion molecules, e.g. integrins. We evaluated the expression of integrins in naturally shed menstrual endometrium and the adhesion pattern of this tissue to different components of the ECM. To identify integrins and matrix components involved, blocking studies were performed. Most of the 15 menstrual tissue samples showed positive staining for each of the integrins investigated, except alpha(4)beta(1). Compared with binding to collagen IV, which was set at 100%, adhesion to collagen I was 93% (not significant), to fibronectin 87% (P < 0.05), and to laminin 74% (P < 0.05). Scanning electron micoscopy showed that endometrium adhered to laminin but hardly spread, whereas spreading was observed when layered on the other coatings. Compared with the control (which was set at 100%), incubation with 4B4, a monoclonal antibody against the integrin beta(1) subunit, showed a significant reduction of adhesion (to approximately 50%; P < 0.05) when layered on laminin and a smaller reduction (to 82-86%; P < 0.05) when layered on the other three coatings. Incubation with antibody GOH3 against integrin alpha(6)beta(1) resulted in a similar reduction in adhesion to laminin. Incubation with an RGD peptide significantly reduced adhesion (to 84%; P < 0.05) when plated on fibronectin. In conclusion, antegradely shed menstrual endometrium expresses various integrins. It shows preferential attachment to collagen IV and collagen I, when compared with fibronectin and laminin. Blockage of the integrin beta(1) subunit resulted in greatest disruption to adhesion when layered on laminin, implying that the interaction was mediated by the alpha(6)beta(1) integrin. Since this adhesion was not completely blocked, other mechanisms are likely to be involved.

We have investigated the adhesion of endometrial tissue isolated from antegradely shed menstrual effluent to amnion and peritoneum. This endometrial tissue was cultured overnight on either side of intact and stripped amnion and on the mesothelial side of peritoneum. Light and electron microscopy were applied to evaluate adhesion. With light microscopy adhesion of endometrial fragments to stripped membranes was observed in nine out of 12 specimens and in 12 out of 13 specimens when layered on the extracellular matrix side of amnion. Adhesion when layered on the epithelial side was seen in only four out of 13 specimens. However, when using scanning electron microscopy adhesion of menstrual endometrial tissue could be visualized in all samples. Numerous adhering fragments were seen when layered on the extracellular matrix side of untreated amnion. On several occasions not only adhesion but also spreading of cells was observed. When layered on the epithelial side of untreated amnion or peritoneum, adhesion was exclusively seen at locations where the epithelium was damaged or absent. These findings were confirmed by transmission electron microscopy. These observations indicate that endometrial tissue isolated from antegradely shed menstrual effluent preferentially adheres to subepithelial structures of amnion and peritoneum. The lack of adhesion to epithelial cells suggests that an intact mesothelial lining prevents adhesion of menstrual endometrial tissue.

Background Hormone replacement therapy increases the risk of developing ulcerative colitis in postmenopausal women. Chronic intestinal inflammation predisposes to colon cancer development, but effects of female hormones on colitis-associated cancer development have not been examined. Aim To investigate the role of female hormones in the dextran sodium sulfate (DSS)-azoxymethane (AOM) mouse model for colitis-associated cancer. Design We performed ovariectomies, or sham operations, on mice, and supplemented these animals with indicated hormones. Additionally, we used oestrogen receptor α or β ( Erα or Erβ ) mutant mice. To study colitis or colitis-associated cancer, we used DSS only, or DSS and AOM, respectively. Results Ovariectomy protects female mice against colitis-associated tumour development. Hormone replacement in ovariectomised mice with either oestradiol (E2), medroxyprogesterone acetate or a combination of both suggests that oestrogens are the ovary-derived factor that promotes tumour development in the context of inflammatory damage. E2-treated animals showed increased clinical symptoms and Il-6 production upon DSS-induced colitis and enhanced epithelial proliferation. Treatment with E2 markedly increased the numbers of polyps in ovariectomised mice and also strongly promoted tumour progression with all E2-treated animals developing at least one invasive adenocarcinoma, whereas, placebo-treated animals developed adenomas only. Using Er mutant mice, we find that the protumorigenic effect of oestrogen depends on both Erα and Erβ. Conclusions Our results suggest that oestrogens promote inflammation-associated cancer development by impairing the mucosal response to inflammatory damage.

It recently has been recognized that men develop colonic adenomas and carcinomas at an earlier age and at a higher rate than women. In the Apc(Pirc/+) (Pirc) rat model of early colonic cancer, this sex susceptibility was recapitulated, with male Pirc rats developing twice as many adenomas as females. Analysis of large datasets revealed that the Apc(Min/+) mouse also shows enhanced male susceptibility to adenomagenesis, but only in the colon. In addition, WT mice treated with injections of the carcinogen azoxymethane (AOM) showed increased numbers of colonic adenomas in males. The mechanism underlying these observations was investigated by manipulation of hormonal status. The preponderance of colonic adenomas in the Pirc rat model allowed a statistically significant investigation in vivo of the mechanism of sex hormone action on the development of colonic adenomas. Females depleted of endogenous hormones by ovariectomy did not exhibit a change in prevalence of adenomas, nor was any effect observed with replacement of one or a combination of female hormones. In contrast, depletion of male hormones by orchidectomy (castration) markedly protected the Pirc rat from adenoma development, whereas supplementation with testosterone reversed that effect. These observations were recapitulated in the AOM mouse model. Androgen receptor was undetectable in the colon or adenomas, making it likely that testosterone acts indirectly on the tumor lineage. Our findings suggest that indirect tumor-promoting effects of testosterone likely explain the disparity between the sexes in the development of colonic adenomas.

A prospective study was performed to determine the effects of the angiostatic compounds anti-hVEGF antibody, TNP-470, endostatin, and anginex on the vascularization and on endometriosis-like lesion formation in the chicken chorioallantoic membrane model. Endometriosis-like lesion formation was significantly impaired after treatment with angiostatic agents, which was associated with decreased vessel densities in the surrounding chorioallantoic membrane and more necrosis in the endometriosis-like lesions.

[This corrects the article DOI: 10.1371/journal.pone.0022620.].

Sampson's transplantation theory for the pathogenesis of peritoneal endometriosis is widely accepted. The events that take place, however, on the cellular and subcellular level during the transition of endometrial tissue in the abdominal cavity into peritoneal endometriosis remain controversial. The mesothelium plays a central role in the debate on this subject. The interaction between endometrium and peritoneum has been studied in an in-vitro model using amnion, peritoneum and mesothelial cells in culture on the one hand and cyclic and menstrual endometrium on the other hand. The results of these studies indicate that (i) an intact mesothelial lining prevents adhesion of shed endometrial tissue, (ii) shed endometrial tissue adheres to the peritoneal extracellular matrix and (iii) menstrual effluent creates its own adhesion sites by damaging the mesothelial lining thus exposing the extracellular matrix. Therefore we conclude that the mesothelium has the properties of Teflon, while the extracellular matrix resembles Velcro.

Olfactomedin-4 (OLFM-4) is an extracellular matrix protein that is highly expressed in human endometrium. We have examined the regulation and function of OLFM-4 in normal endometrium and in cases of endometriosis and endometrial cancer. OLFM-4 expression levels are highest in proliferative-phase endometrium, and 17 beta-estradiol up-regulates OLFM-4 mRNA in endometrial explant cultures. Using the luciferase reporter under control of the OLFM-4 promoter, it was shown that both 17 beta-estradiol and OH-tamoxifen induce luciferase activity, and epidermal growth factor receptor-1 is required for this estrogenic response. In turn, EGF activates the OLFM-4 promoter, and estrogen receptor-alpha is needed for the complete EGF response. The cellular functions of OLFM-4 were examined by its expression in OLFM-4-negative HEK-293 cells, which resulted in decreased vimentin expression and cell adherence as well as increased apoptosis resistance. In cases of endometriosis and endometrial cancer, OLFM-4 expression correlated with the presence of epidermal growth factor receptor-1 and estrogen receptor-alpha (or estrogen signaling). An increase of OLFM-4 mRNA was observed in the endometrium of endometriosis patients. No change in OLFM-4 expression levels were observed in patients with endometrial cancer relative with controts. In conclusion, cross-talk between estrogen and EGF signaling regulates OLFM-4 expression. The role of OLFM-4 in endometrial tissue remodeling before the secretory phase and during the predisposition and early events in endometriosis can be postulated but requires additional investigation. (Am J Pathol 2010, 177:2495-2508: DOI: 10.2353/ajpath.2010.100026

We characterised the effects of selective oestrogen receptor modulators (SERM) in explant cultures of human endometrium tissue. Endometrium tissues were cultured for 24 h in Millicell-CM culture inserts in serum-free medium in the presence of vehicle,17 beta-estradiol (17 beta-E2,1 nM), oestrogen receptor (ER) antagonist ICI 164.384 (40 nM), and 4-OH-tamoxifen (40 nM), raloxifene (4 nM), lasofoxifene (4 nM)and acolbifene (4 nM). Protein expression of ER alpha, ER beta 1 and Ki-67 were evaluated by immunohistochemistry (IHC). The proliferative fraction was assessed by counting the number of Ki-67 positive cells. Nuclear staining of ER( and ER(1 was observed in the glandular epithelium and stroma of pre- and postmenopausal endometrium. ER(1 protein was also localized in the endothelial cells of blood vessels. Treating premenopausal endometrium tissue with 17 beta-E2 increased the fraction of Ki-67 positive cells (p < 0.001) by 55% in glands compared to the control. Raloxifene (4 nM) increased (p < 0.05) the Ki-67 positive fraction. All other SERMS did not affect proliferation in this model. Treating postmenopausal endometrium with 17(-E2 increased (p < 0.001) the fraction of Ki-67 positive cells by 250% in glands compared to the control. A similar effect was also seen for 4-OH-tamoxifen, whereas the rest of SERMs did not stimulate proliferation. We demonstrated that oestradiol increases the fraction of proliferating cells in short term explant cultures of postmenopausal endometrium. In addition, we were able to reveal the agonistic properties of 4-OH-tamoxifen and confirm that raloxifene and next-generation SERMs acolbifene and lasofoxifene were neutral on the human postmenopausal endometrium. (C) 2008 Elsevier Ltd. All rights reserved.

Fundamental and genetic differences between women in the endometrium may cause some to develop endometriosis, whereas others do not. Oral contraceptives (OC) may have an effect on the endometrium, rendering the development of endometriosis less likely.Endometrium from women using OC (OCE) and menstrual endometrium (ME) from normal cycling women were transplanted onto the chicken chorioallantoic membrane (CAM), and endometriosis-like lesion formation was evaluated. Microarray gene expression profiling was performed to identify differentially expressed genes in the endometrium from these groups. Microarray data were validated by real-time PCR.Less endometriosis-like lesions were formed after transplantation of OCE than after transplantation of ME (p.05). Most of the differentially expressed genes belong to the TGFbeta superfamily. Real-time PCR validation revealed that inhibin betaA (INHBA) expression was significantly decreased in OCE as compared to ME.OC use affects the characteristics of endometrium, rendering it less potent to develop into endometriosis.

To identify specific markers of rectovaginal endometriotic nodule vasculature, highly enriched preparations of vascular endothelial cells and pericytes were obtained from endometriotic nodules and control endometrial and myometrial tissue by laser capture microdissection (LCM), and gene expression profiles were screened by microarray analysis. Of the 18 400 transcripts on the arrays, 734 were significantly overexpressed in vessels from fibromuscular tissue and 923 in vessels from stromal tissue of endometriotic nodules, compared with vessels dissected from control tissues. The most frequently expressed transcripts included known endothelial cell-associated genes, as well as transcripts with little or no previous association with vascular cells. The higher expression in blood vessels was further corroborated by immunohistochemical staining of six potential markers, five of which showed strong expression in pericytes. The most promising marker was matrix Gla protein, which was found to be present in both glandular epithelial cells and vascular endothelial cells of endometriotic lesions, although it was barely expressed at all in normal endometrium. LCM, combined with microarray analysis, constitutes a powerful tool for mapping the transcriptome of vascular cells. After immunohistochemical validation, markers of vascular endothelial and perivascular cells from endometriotic nodules could be identified, which may provide targets to improve early diagnosis or to selectively deliver therapeutic agents.

Deep infiltrating endometriosis is characterized by the presence of nodular lesions largely composed of fibromuscular tissue. Transforming growth factor beta 1 (TGF-beta1) is the cytokine most causatively associated with disorders characterized by fibrosis throughout the body. Therefore, the hypothesis was tested that mechanisms increasing the fraction of biologically active TGF-beta1, such as TGF-beta 1 gene polymorphisms, lead to an increased risk of developing deep infiltrating endometriosis. The frequency of the -509C/T polymorphism of the TGF-beta 1 gene was tested in women with deep infiltrating endometriosis (n = 72), gynecological patients without symptoms of endometriosis (n = 95) and healthy females (n = 93). Detection of the -509C/T polymorphisms was performed using PCR-restriction fragment length polymorphism analysis. We did not observe statistically significant differences in the frequency of the -509C/T polymorphism between the groups. Our study does not support an association between the -509C/T polymorphism of the TGF-beta 1 gene and an increased risk of deep infiltrating endometriosis.

Disorders of estrogen-responsive tissues are frequently associated with aberrations in steroid metabolism due to altered expression of synthesizing and metabolizing enzymes. For instance, overexposure to unopposed 17beta-estradiol has been associated with the pathogenesis of endometrial proliferative disorders, such as endometriosis. Investigations into the metabolic conversion in tissues and cells have been rather limited. This is mostly due to fact that such studies have to make use of radioactive steroid hormones and expensive equipment to obtain sufficient sensitivity. We adapted a sensitive non-radioactive HPLC method to study estrogen metabolism in more detail. This HPLC method is based on the solid phase extraction of estrogens and the derivatization of the steroids with 2-(4-carboxy-phenyl)-5,6-dimethylbenzimidazole. The technique is sensitive, robust and is useful for the detection of aromatase, 17beta-HSD types 1 and 2 and sulfatase activities in lysates of placenta and endometrium.

Genomic profiling was performed on explants of late proliferative phase human endometrium after 24-h treatment with progesterone (P) or oestradiol and progesterone (17 beta-E-2+P) and on explants of menstrual phase endometrium treated with 17 beta-E-2+P. Gene expression was validated with real-time PCR in the samples used for the arrays, in endometrium collected from early and mid-secretory phase endometrium, and in additional experiments performed on new samples collected in the menstrual and late proliferative phase. The results show that late proliferative phase human endometrium is more responsive to progestins than menstrual phase endometrium, that the expression of several genes associated with embryo implantation (i.e. thrombomodulin, monoamine oxidase A, SPARC-like 1) can be induced by P in vitro, and that genes that are fully dependent on the continuous presence of 17 beta-E-2 during P exposure can be distinguished from those that are P-dependent to a lesser extent. Therefore, 17 beta-E-2 selectively primes implantation-related genes for the effects of P.

SYD985 is a HER2-targeting ADC based on trastuzumab and vc-seco-DUBA, Synthon's proprietary cleavable linker-duocarmycin payload. Vc-seco-DUBA was coupled to cysteines after partial reduction on the interchain disulfides of trastuzumab. SYD985 was obtained after purification by Hydrophobic Interaction Chromatography to yield a well-defined ADC consisting of predominantly DAR 2 and DAR 4 species. To evaluate the therapeutic potential of this new ADC, and to prepare clinical development, mechanistic in vitro studies and in vivo PDX studies were conducted to compare SYD985 head-to-head to T-DM1 (Kadcyla®), another trastuzumab-based ADC with a toxin of the maytansinoid class in combination with a non-cleavable linker. SYD985 and T-DM1 had similar binding-affinities to HER2 and showed similar internalization. In vitro cytotoxicity assays showed similar potencies and efficacies in HER2 3+ cell lines, but in cell lines with low HER2 expression, SYD985 was 3 to 50-fold more potent than T-DM1. In contrast to T-DM1, SYD985 efficiently induced bystander killing in vitro of HER2 negative (HER2 0) cells when mixed with HER2 3+, 2+, or 1+ cells. SYD985 efficiently killed HER2 0 cells, even in the presence of only 20% of HER2 3+ cells. At pH conditions relevant for tumors, cathepsin B cleavage studies showed efficient release of the active toxin from SYD985 but not from T-DM1. These in vitro data suggest that SYD985 might be a more potent ADC in HER2 expressing tumors in vivo, allowing the treatment of tumor tissues with low or heterogeneous membrane expression of HER2. In line with this, in vivo anti-tumor studies in breast cancer PDX models showed that SYD985 is very active in HER2 3+, 2+, and 1+ models whereas T-DM1 only showed significant anti-tumor activity in HER2 3+ breast cancer PDX models. We conclude that the properties of SYD985 may enable expansion of the target population to patients who have low HER2 expressing breast cancer, a patient population with unmet high medical need. Early phase clinical evaluation with SYD985 is ongoing. Citation Format: Willem Dokter, Miranda van der Lee, Patrick Groothuis, Ruud Ubink, Monique van der Vleuten, Tanja van Achterberg, Eline Loosveld, Desirée Damming, Myrthe Rouwette, David Egging, Diels van den Dobbelsteen, Patrick Beusker, peter goedings, Gijs Verheijden, Jacques Lemmens, Marco Timmers. The preclinical profile of the duocarmycin-based HER2-targeting ADC SYD985 predicts for clinical benefit in low HER2-expressing breast cancers. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5360. doi:10.1158/1538-7445.AM2015-5360

Background: Aberrations in mediators of Ras signaling may increase the risk of developing recurrent endometrial carcinoma. Patients and methods: Primary tumors of patients with (n = 44) and without (n = 44) recurrent stage I endometrioid endometrial carcinoma were compared regarding the presence of K-ras mutations (codons 12 and 13), B-raf mutations (V599), and RASSF1A gene promoter methylation. Results: K-ras mutations were present in 18% of the patients independent of recurrent disease. No B-raf mutations were found. RASSF1A methylation was demonstrated in 85% of endometrial carcinomas, independent of recurrence. The presence of K-ras mutations and RASSF1A promoter methylation were not related, either directly or inversely. Analysis in premenopausal endometrial carcinomas demonstrated K-ras mutations in 40%, no B-raf mutations, and RASSF1A promoter methylation in 70% of the cases. RASSF1A methylation was also observed in samples of cyclic (n = 14), hyperplastic (n = 8), and atrophic (n = 13) endometrial tissues in 21%, 50% and 38%, respectively. Conclusions:RASSF1A methylation was observed in a high frequency in endometrioid endometrial carcinoma whereas K-ras and B-raf mutations were observed in a low frequency. No association was observed with the development of recurrent disease. High-frequency RASSF1A methylation in premenopausal carcinomas and an increased frequency in endometrial hyperplasia indicate that this may be an early event in endometrial carcinogenesis.