Epigenetic Regulation of Phenotypic Sexual Plasticity Inducing Skewed Sex Ratio in Zebrafish.

The plasticity of sexual phenotype in response to environmental conditions results in biased sex ratios, and their variation has an effect on population dynamics. Epigenetic modifications can modulate sex ratio variation in species, where sex is determined by genetic and environmental factors. However, the role of epigenetic mechanisms underlying skewed sex ratios is far from being clear and is still an object of debate in evolutionary developmental biology. In this study, we used zebrafish as a model animal to investigate the effect of DNA methylation on sex ratio variation in sex-biased families in response to environmental temperature. Two sex-biased families with a significant difference in sex ratio were selected for genome-wide DNA methylation analysis using reduced representation bisulfite sequencing (RRBS). The results showed significant genome-wide methylation differences between male-biased and female-biased families, with a greater number of methylated CpG sites in testes than ovaries. Likewise, pronounced differences between testes and ovaries were identified within both families, where the male-biased family exhibited a higher number of methylated sites than the female-biased family. The effect of temperature showed more methylated positions in the high incubation temperature than the control temperature. We found differential methylation of many reproduction-related genes (e.g., sox9a, nr5a2, lhx8a, gata4) and genes involved in epigenetic mechanisms (e.g., dnmt3bb.1, dimt1l, hdac11, h1m) in both families. We conclude that epigenetic modifications can influence the sex ratio variation in zebrafish families and may generate skewed sex ratios, which could have a negative consequence for population fitness in species with genotype-environment interaction sex-determining system under rapid environmental changes.

α/ß-Hydrolase D16B Truncation Results in Premature Sperm Capacitation in Cattle.

Recently it was shown that a specific form of male infertility in Holstein cattle was caused by a nonsense variant in the α/ß-hydrolase domain-containing 16B (ABHD16B) gene resulting in a protein truncation at amino acid position 218 (p.218Q*) and loss of function. Lipidomics showed that the absence of ABHD16B influenced the content of phosphatidylcholine (PC), ceramide (Cer), diacylglycerol (DAG), and sphingomyelin (SM) in variant carrier sperm membranes. However, the exact cause of infertility in affected sires has remained unclear until now. To elucidate the cause of infertility, we analyzed (i) standard sperm parameters (i.e., total sperm number, morphological intact sperm, total sperm motility), (ii) in vitro fertilizability and effects on early embryonic development, and (iii) sperm survival rates (i.e., capacitation time). The affected spermatozoa showed no changes in the usual sperm parameters and were also capable of fertilization in vitro. Furthermore, the absence of ABHD16B did not affect early embryonic development. Based on these results, it was concluded that the affected spermatozoa appeared to be fertilizable per se. Consequently, the actual cause of the inability to fertilize could only be due to a time- and/or place-dependent process after artificial insemination and before fertilization. A process fundamental to the ability to fertilize after insemination is capacitation. Capacitation is a biochemical maturation process that spermatozoa undergo in the female genital tract and is inevitable for the successful fertilization of the oocyte. It is known that the presence and concentration of certain sperm membrane lipids are essential for the correct course of capacitation. However, precisely these lipids are absent in the membrane of spermatozoa affected by the ABHD16B truncation. Since all other causes of fertilization inability were excluded in the previous experiments, consequently, the only remaining hypothesis was that the loss of function of ABHD16B leads to a capacitation disruption. We were able to show that heterozygous and homozygous affected spermatozoa exhibit premature capacitation and therefore decay before fertilization. This effect of the loss of function of ABHD16B has not been described before and our studies now revealed why sires harboring the variant in the ABHD16B gene are infertile.

Evaluation of circulating microRNAs as non-invasive biomarkers in the diagnosis of ovarian cancer: a case-control study.

PURPOSE: Ovarian cancer is the seventh most frequent form of malignant diseases in women worldwide and over 150,000 women die from it every year. More than 70 percent of all ovarian cancer patients are diagnosed at a late-stage disease with poor prognosis necessitating the development of sufficient screening biomarkers. MicroRNAs displayed promising potential as early diagnostics in various malignant diseases including ovarian cancer. The presented study aimed at identifying single microRNAs and microRNA combinations detecting ovarian cancer in vitro and in vivo. METHODS: Intracellular, extracellular and urinary microRNA expression levels of twelve microRNAs (let-7a, let-7d, miR-10a, miR-15a, miR-15b, miR-19b, miR-20a, miR-21, miR-100, miR-125b, miR-155, miR-222) were quantified performing quantitative real-time-PCR. Therefore, the three ovarian cancer cell lines SK-OV-3, OAW-42, EFO-27 as well as urine samples of ovarian cancer patients and healthy controls were analyzed. RESULTS: MiR-15a, miR-20a and miR-222 showed expression level alterations extracellularly, whereas miR-125b did intracellularly across the analyzed cell lines. MicroRNA expression alterations in single cell lines suggest subtype specificity in both compartments. Hypoxia and acidosis showed scarce effects on single miRNA expression levels only. Furthermore, we were able to demonstrate the feasibility to clearly detect the 12 miRNAs in urine samples. In urine, miR-15a was upregulated whereas let-7a was down-regulated in ovarian cancer patients. CONCLUSION: Intracellular, extracellular and urinary microRNA expression alterations emphasize their great potential as biomarkers in liquid biopsies. Especially, miR-15a and let-7a qualify for possible circulating biomarkers in liquid biopsies of ovarian cancer patients.

Anticarcinogenic Effects of Odorant Substances Citral, Citrathal R and Cyclovertal on Breast Cancer in vitro.

PURPOSE: In 2020, breast cancer still represents the most common type of cancer in women worldwide. Depending on the specific molecular subtype, clinical breast cancer management comprises surgery, radiotherapy, chemotherapy and targeted therapy. Furthermore, there are some therapeutic approaches from the field of complementary and alternative medicine. Current research focuses on the elucidation of new therapeutic targets for treatment development. Odorant substances affect apoptosis, proliferation and cell cycle in healthy and cancerous cells. Exact signalling pathways involved are not entirely clear. The present study aims to analyse their therapeutic potential in breast cancer. METHODS: This study focuses on the effect of commonly used odorant substances (citral, citrathal R, cyclovertal, para-cymol, hexylacetat, herbavert, dihydromyrcerol and limonen) on the breast cancer cell lines MDA-MB-231, T47-D and BT474. Methodologically, this study applied cell culturing, MTT assay for detection of IC50 of the odorant substance, RNA purification followed by qRT-PCR, protein isolation and Western Blot, as well as immunocytochemistry. Further, this study investigates the role of transient receptor potential channel V1 (TRPV1), involved in the mechanisms of action for some odorant substances. Therefore, capsazepine, a TRPV1 antagonist, was used. RESULTS: The odorant substances citral, citrathal R and cyclovertal have significant pro-apoptotic (p < 0.001), anti-proliferative (p < 0.001) and cell cycle-arresting effects measurable in RNA expression as well as in protein levels and immunocytochemical staining. The combination of citral and capsazepine no longer showed significant pro-apoptotic, antiproliferative, and cell cycle inhibitory effects compared to the compounds alone. This indicates that TRPV1 is necessary for the signal transduction of citral. CONCLUSION: This present study reveals three odorant substances with effects on cell viability, indicating their potential use in breast cancer therapy.

Sperm Lipid Markers of Male Fertility in Mammals.

Sperm plasma membrane lipids are essential for the function and integrity of mammalian spermatozoa. Various lipid types are involved in each key step within the fertilization process in their own yet coordinated way. The balance between lipid metabolism is tightly regulated to ensure physiological cellular processes, especially referring to crucial steps such as sperm motility, capacitation, acrosome reaction or fusion. At the same time, it has been shown that male reproductive function depends on the homeostasis of sperm lipids. Here, we review the effects of phospholipid, neutral lipid and glycolipid homeostasis on sperm fertilization function and male fertility in mammals.

Detection of microRNA in urine to identify patients with endometrial cancer: a feasibility study.

OBJECTIVE: To find dysregulated urinary microRNAs associated with endometrial cancer as a first step in finding a non-invasive new diagnostic biomarker. The second objective is to determine the correlation of urinary microRNAs with clinicopathological characteristics. METHODS: A prospective cohort study of patients presenting with abnormal bleeding between March and November 2019 was performed at the Royal Cornwall Hospital Trust Truro. Urine samples were obtained from women diagnosed with endometrial cancer and benign endometrial sampling. MicroRNA was isolated and quantitative real time PCR was used to detect expression levels of microRNAs. RESULTS: A total of 61 women were included in this study: 24 endometrial cancer patients, and 37 controls. Median age was 64 years (range 45-94) and median body mass index was 29 kg/m2 (range 17-54). MiR-223 was significantly up-regulated in urine of endometrial cancers patients (p=0.003). Furthermore, let7-i, miR-34a, and miR-200c were significantly down-regulated and miR-424 was up-regulated in obese women. In addition, miR-148a and miR-222 were significantly down-regulated in elderly women, and miR-16, miR-26b, and miR-200c were significantly deregulated in women with multiple comorbidities. CONCLUSION: MicroRNA expression levels in urine can potentially be used as a non-invasive diagnostic test for endometrial cancer. Furthermore, aberrant microRNA expression in urine is associated with patient characteristics. Further research in larger trials is needed to validate the potential utility of urinary microRNAs.

Usefulness of microRNA detection in the diagnostics of endometrial cancer.

INTRODUCTION: MicroRNAs (miRNAs) are noncoding RNAs that regulate gene expression and contribute to the development of cancer. They have been shown to be stable in tissue samples and may be promising diagnostic biomarkers for endometrial cancer. MATERIAL AND METHODS: A retrospective cohort study of women diagnosed with endometrial cancer between January 2017 and December 2017 was performed at the Royal Cornwall Hospital. Archived formalin-fixed paraffin-embedded samples were obtained from patients with endometrial cancer and healthy women. MicroRNA was isolated and quantitative real-time polymerase chain reaction was used to detect expression levels of miRNAs. RESULTS: A total of 76 women were included: 36 endometrial cancer patients, 40 healthy controls. A distinct panel of miR-200a, miR-200b, miR-200c, miR-205, and miR-182 showed an area under the curve of 0.958, sensitivity 92%, specificity 89%, positive predictive value of 89% (95% CI 82%-94%) and negative predictive value of 91% (95% CI 85%-96%) in diagnosing endometrial cancer. High miR-182 expression levels were significantly related to high-grade endometrioid tumors compared with low-grade tumors. CONCLUSIONS: We demonstrated high diagnostic accuracy of miRNA for detecting endometrial cancer. In addition, miRNA contributed to an improvement in distinguishing between high-grade and low-grade endometrioid tumors.

The cutoff for estrogen and progesterone receptor expression in endometrial cancer revisited: a European Network for Individualized Treatment of Endometrial Cancer collaboration study.

There is no consensus on the cutoff for positivity of estrogen receptor (ER) and progesterone receptor (PR) in endometrial cancer (EC). Therefore, we determined the cutoff value for ER and PR expression with the strongest prognostic impact on the outcome. Immunohistochemical expression of ER and PR was scored as a percentage of positive EC cell nuclei. Cutoff values were related to disease-specific survival (DSS) and disease-free survival (DFS) using sensitivity, specificity, and multivariable regression analysis. The results were validated in an independent cohort. The study cohort (n = 527) included 82% of grade 1-2 and 18% of grade 3 EC. Specificity for DSS and DFS was highest for the cutoff values of 1-30%. Sensitivity was highest for the cutoff values of 80-90%. ER and PR expression were independent markers for DSS at cutoff values of 10% and 80%. Consequently, three subgroups with distinct clinical outcomes were identified: 0-10% of ER/PR expression with, unfavorable outcome (5-year DSS = 75.9-83.3%); 20-80% of ER/PR expression with, intermediate outcome (5-year DSS = 93.0-93.9%); and 90-100% of ER/PR expression with, favorable outcome (5-year DSS = 97.8-100%). The association between ER/PR subgroups and outcomes was confirmed in the validation cohort (n = 265). We propose classification of ER and PR expression based on a high-risk (0-10%), intermediate-risk (20-80%), and low-risk (90-100%) group.

Frameshift Variant in Novel Adenosine-A1-Receptor Homolog Associated With Bovine Spastic Syndrome/Late-Onset Bovine Spastic Paresis in Holstein Sires.

Since their first description almost 100 years ago, bovine spastic paresis (BSP) and bovine spastic syndrome (BSS) are assumed to be inherited neuronal-progressive diseases in cattle. Affected animals are characterized by (frequent) spasms primarily located in the hind limbs, accompanied by severe pain symptoms and reduced vigor, thus initiating premature slaughter or euthanasia. Due to the late onset of BSP and BSS and the massively decreased lifespan of modern cattle, the importance of these diseases is underestimated. In the present study, BSP/BSS-affected German Holstein breeding sires from artificial insemination centers were collected and pedigree analysis, genome-wide association studies, whole genome resequencing, protein-protein interaction network analysis, and protein-homology modeling were performed to elucidate the genetic background. The analysis of 46 affected and 213 control cattle revealed four significantly associated positions on chromosome 15 (BTA15), i.e., AC_000172.1:g.83465449A>G (-log10P = 19.17), AC_000172.1:g.81871849C>T (-log10P = 8.31), AC_000172.1:g.81872621A>T (-log10P = 6.81), and AC_000172.1:g.81872661G>C (-log10P = 6.42). Two additional loci were significantly associated located on BTA8 and BTA19, i.e., AC_000165.1:g.71177788T>C and AC_000176.1:g.30140977T>G, respectively. Whole genome resequencing of five affected individuals and six unaffected relatives (two fathers, two mothers, a half sibling, and a full sibling) belonging to three different not directly related families was performed. After filtering, a homozygous loss of function variant was identified in the affected cattle, causing a frameshift in the so far unknown gene locus LOC100848076 encoding an adenosine-A1-receptor homolog. An allele frequency of the variant of 0.74 was determined in 3,093 samples of the 1000 Bull Genomes Project.

Mutually distinguishing microRNA signatures of breast, ovarian and endometrial cancers in vitro.

Early diagnosis and therapy in the first stages of a malignant disease is the most crucial factor for successful cancer treatment and recovery. Currently, there is a high demand for novel diagnostic tools that indicate neoplasms in the first or pre­malignant stages. MicroRNAs (miRNA or miR) are small non­coding RNAs that may act as oncogenes and downregulate tumor­suppressor genes. The detection and mutual discrimination of the three common female malignant neoplasia types breast (BC), ovarian (OC) and endometrial cancer (EC) could be enabled by identification of tumor entity­specific miRNA expression differences. In the present study, the relative expression levels of 25 BC, EC and OC­related miRNAs were assessed by reverse transcription­quantitative PCR and determined using the 2­ΔΔCq method for normalization against the mean of four housekeeping genes. Expression levels of all miRNAs were analyzed by regression against cell line as a factor. An expression level­based discrimination between BC and OC cell types was obtained for a subgroup of ten different miRNA types. miR­30 family genes, as well as three other miRNAs, were found to be uniformly upregulated in OC cells compared with BC cells. BC and EC cells could be distinguished by the expression profiles of six specific miRNAs. In addition, four miRNAs were differentially expressed between EC and OC cells. In conclusion, miRNAs were identified as a potential novel tool to detect and mutually discriminate between BC, OC and EC. Based on a subset of 25 clinically relevant human miRNA types, the present study could significantly discriminate between these three female cancer types by means of their expression levels. For further verification and validation of miRNA­based biomarker expression signatures that enable valuable tumor detection and characterization in routine screening or potential therapy monitoring, additional and extended in vitro analyses, followed by translational studies utilizing patients' tissue and liquid biopsy materials, are required.

Endoxifen and fulvestrant regulate estrogen-receptor α and related DEADbox proteins.

Breast cancer (BC) represents the most common type of cancer in females worldwide. Endocrine therapy evolved as one of the main concepts in treatment of hormone-receptor positive BC. Current research focuses on the elucidation of tumour resistance mechanisms against endocrine therapy. In a translational in vitro approach, potential regulatory effects of clinically implemented BC anti-oestrogens on ERα, its coactivators DDX5, DDX17 and other DEADbox proteins as well as on the proliferation markers cyclin D1 and Ki67 were investigated on both the RNA and protein level. BC in vitro models for hormone-receptor positive (MCF-7, T-47D) and hormone-receptor negative cells (BT-20) were subjected to endocrine therapy. Anti-oestrogen-dependent expression regulation of target genes on the transcriptional and translational level was quantified and statistically assessed. Endocrine therapy decreases the expression levels of Ki67, cyclin D1 and ERα in hormone-receptor positive cells. In the hormone-receptor negative cells, the three parameters remained stable after endocrine therapy. Endoxifen triggers a downregulation of DDX5 and DDX23 in MCF-7 cells. Fulvestrant treatment downregulates the expression levels of all investigated DEADbox proteins in MCF-7 cells. In T-47D cells, endoxifen and fulvestrant lead to a decrease of all target gene expression levels. Interestingly, endocrine therapy affects DEADbox RNA expression levels in BT-20 cells, too. However, this result could only be confirmed for DDX1, immunocytologically. The investigated DEADbox proteins appear to correlate with the oestrogen-dependent tumourigenesis in hormone-receptor positive BC and show expression alterations after endocrine treatment.

Preoperative risk stratification in endometrial cancer (ENDORISK) by a Bayesian network model: A development and validation study.

BACKGROUND: Bayesian networks (BNs) are machine-learning-based computational models that visualize causal relationships and provide insight into the processes underlying disease progression, closely resembling clinical decision-making. Preoperative identification of patients at risk for lymph node metastasis (LNM) is challenging in endometrial cancer, and although several biomarkers are related to LNM, none of them are incorporated in clinical practice. The aim of this study was to develop and externally validate a preoperative BN to predict LNM and outcome in endometrial cancer patients. METHODS AND FINDINGS: Within the European Network for Individualized Treatment of Endometrial Cancer (ENITEC), we performed a retrospective multicenter cohort study including 763 patients, median age 65 years (interquartile range [IQR] 58-71), surgically treated for endometrial cancer between February 1995 and August 2013 at one of the 10 participating European hospitals. A BN was developed using score-based machine learning in addition to expert knowledge. Our main outcome measures were LNM and 5-year disease-specific survival (DSS). Preoperative clinical, histopathological, and molecular biomarkers were included in the network. External validation was performed using 2 prospective study cohorts: the Molecular Markers in Treatment in Endometrial Cancer (MoMaTEC) study cohort, including 446 Norwegian patients, median age 64 years (IQR 59-74), treated between May 2001 and 2010; and the PIpelle Prospective ENDOmetrial carcinoma (PIPENDO) study cohort, including 384 Dutch patients, median age 66 years (IQR 60-73), treated between September 2011 and December 2013. A BN called ENDORISK (preoperative risk stratification in endometrial cancer) was developed including the following predictors: preoperative tumor grade; immunohistochemical expression of estrogen receptor (ER), progesterone receptor (PR), p53, and L1 cell adhesion molecule (L1CAM); cancer antigen 125 serum level; thrombocyte count; imaging results on lymphadenopathy; and cervical cytology. In the MoMaTEC cohort, the area under the curve (AUC) was 0.82 (95% confidence interval [CI] 0.76-0.88) for LNM and 0.82 (95% CI 0.77-0.87) for 5-year DSS. In the PIPENDO cohort, the AUC for 5-year DSS was 0.84 (95% CI 0.78-0.90). The network was well-calibrated. In the MoMaTEC cohort, 249 patients (55.8%) were classified with <5% risk of LNM, with a false-negative rate of 1.6%. A limitation of the study is the use of imputation to correct for missing predictor variables in the development cohort and the retrospective study design. CONCLUSIONS: In this study, we illustrated how BNs can be used for individualizing clinical decision-making in oncology by incorporating easily accessible and multimodal biomarkers. The network shows the complex interactions underlying the carcinogenetic process of endometrial cancer by its graphical representation. A prospective feasibility study will be needed prior to implementation in the clinic.

Urinary Exosomal MicroRNAs as Potential Non-invasive Biomarkers in Breast Cancer Detection.

INTRODUCTION: Breast cancer (BC) is the most frequent malignant disease in women worldwide and is therefore challenging for the healthcare system. Early BC detection remains a leading factor that improves overall outcome and disease management. Aside from established screening procedures, there is a constant demand for additional BC detection methods. Routine BC screening via non-invasive liquid biopsy biomarkers is one auspicious approach to either complete or even replace the current state-of-the-art diagnostics. The study explores the diagnostic potential of urinary exosomal microRNAs with specific BC biomarker characteristics to initiate the potential prospective application of non-invasive BC screening as routine practice. METHODS: Based on a case-control study (69 BC vs. 40 healthy controls), expression level quantification and subsequent biostatistical computation of 13 urine-derived microRNAs were performed to evaluate their diagnostic relevance in BC. RESULTS: Multilateral statistical assessment determined and repeatedly confirmed a specific panel of four urinary microRNA types (miR-424, miR-423, miR-660, and let7-i) as a highly specific combinatory biomarker tool discriminating BC patients from healthy controls, with 98.6% sensitivity and 100% specificity. DISCUSSION: Urine-based BC diagnosis may be achieved through the analysis of distinct microRNA panels with proven biomarker abilities. Subject to further validation, the implementation of urinary BC detection in routine screening offers a promising non-invasive alternative in women's healthcare.

Discovery of potential serum and urine-based microRNA as minimally-invasive biomarkers for breast and gynecological cancer.

BACKGROUND: Deregulated microRNAs (miRNAs) in breast and gynecological cancer might contribute to improve early detection of female malignancies. OBJECTIVE: Specification of miRNA types in serum and urine as minimally-invasive biomarkers for breast (BC), endometrial (EC) and ovarian cancer (OC). METHODS: In a discovery phase, serum and urine samples from 17 BC, five EC and five OC patients vs. ten healthy controls (CTRL) were analyzed with Agilent human miRNA microarray chip. Selected miRNA types were further investigated by RT-qPCR in serum (31 BC, 13 EC, 15 OC patients, 32 CTRL) and urine (25 BC, 10 EC, 10 OC patients, 30 CTRL) applying two-sample t-tests. RESULTS: Several miRNA biomarker candidates exhibited diagnostic features due to distinctive expression levels (serum: 26; urine: 22). Among these, miR-518b, -4719 and -6757-3p were found specifically deregulated in BC serum. Four, non-entity-specific, novel biomarker candidates with unknown functional roles were identified in urine (miR-3973; -4426; -5089-5p and -6841). RT-qPCR identified miR-484/-23a (all p⩽ 0.001) in serum as potential diagnostic markers for EC and OC while miR-23a may also serve as an endogenous control in BC diagnosis. CONCLUSIONS: Promising miRNAs as liquid biopsy-based tools in the detection of BC, EC and OC qualified for external validation in larger cohorts.

Association of α/ß-Hydrolase D16B with Bovine Conception Rate and Sperm Plasma Membrane Lipid Composition.

We have identified a Holstein sire named Tarantino who had been approved for artificial insemination that is based on normal semen characteristics (i.e., morphology, thermoresistance, motility, sperm concentration), but had no progeny after 412 first inseminations, resulting in a non-return rate (NRdev) of -29. Using whole genome association analysis and next generation sequencing, an associated nonsense variant in the α/ß-hydrolase domain-containing 16B gene (ABHD16B) on bovine chromosome 13 was identified. The frequency of the mutant allele in the German Holstein population was determined to be 0.0018 in 222,645 investigated cattle specimens. The mutant allele was traced back to Whirlhill Kingpin (bornFeb. 13th, 1959) as potential founder. The expression of ABHD16B was detected by Western blotting and immunohistochemistry in testis and epididymis of control bulls. A lipidome comparison of the plasma membrane of fresh semen from carriers and controls showed significant differences in the concentration of phosphatidylcholine (PC), diacylglycerol (DAG), ceramide (Cer), sphingomyelin (SM), and phosphatidylcholine (-ether) (PC O-), indicating that ABHD16B plays a role in lipid biosynthesis. The altered lipid contents may explain the reduced fertilization ability of mutated sperms.

Osteogenesis imperfecta in a male holstein calf associated with a possible oligogenic origin.

Background: Neuromusculoskeletal anomalies generally in combination with severe clinical symptoms, comprise a heterogeneous group of fairly common and mostly fatal disorders in man and animals. Osteogenesis imperfecta (OI), also known as brittle bone disease, causes bone fragility and deformity. Prominent extra-skeletal accessory manifestations of OI comprise blue/gray sclerae, hearing impairment, lung abnormalities and hypercalciuria. Cases of OI in cattle have been reported. However, no causative mutations have been identified in cattle so far.Aim: To report a possible oligogenic origin identified in a calf from clinically healthy parents suffering from OI.Materials and Methods: A neonatal embryo transfer male Holstein calf developing multiple fractures with bone tissue showing marked osteopenia was used for whole genome re-sequencing as well as its parents. In addition, 2,612 randomly chosen healthy Holstein cattle were genotyped as well as controls.Results: Sixteen candidate genes with potential protein-altering variants were selected revealing non-synonymous variants only within IFITM5 and CRTAP genes. However, in-depth gene analysis did not result in the identification of a single causative mutation in the OI calf.Conclusion: The analysis of the OI case revealed a possible oligogenic origin of the disease attributable to additive effects of three candidate genes, i.e., ABCA13, QRFPR, and IFTIM5.Clinical relevance: Most OI cases in humans and domestic animals reported so far are caused by distinct dominant or recessive monogenic mutations, therefore a potential oligogenic additive genetic effect is a novel finding. Furthermore, the case presented here demonstrates that cross-species genetic analyses might not always be straightforward.

Circulating non­coding RNA­biomarker potential in neoadjuvant chemotherapy of triple negative breast cancer?

Due to the positive association between neoadjuvant chemotherapy (NACT) and the promising early response rates of patients with triple negative breast cancer (TNBC), including probabilities of pathological complete response, NACT is increasingly used in TNBC management. Liquid biopsy­based biomarkers with the power to diagnose the early response to NACT may support established monitoring tools, which are to a certain extent imprecise and costly. Simple serum­ or urine­based analyses of non­coding RNA (ncRNA) expression may allow for fast, minimally­invasive testing and timely adjustment of the therapy regimen. The present study investigated breast cancer­related ncRNAs [microRNA (miR)­7, ­9, ­15a, ­17, ­18a, ­19b, ­21, ­30b, ­222 and ­320c, PIWI­interacting RNA­36743 and GlyCCC2] in triple positive BT­474 cells and three TNBC cell lines (BT­20, HS­578T and MDA­MB­231) treated with various chemotherapeutic agents using reverse transcription­quantitative PCR. Intracellular and secreted microvesicular ncRNA expression levels were analysed using a multivariable statistical regression analysis. Chemotherapy­driven effects were investigated by analysing cell cycle determinants at the mRNA and protein levels. Serum and urine specimens from 8 patients with TNBC were compared with 10 healthy females using two­sample t­tests. Samples from the patients with TNBC were compared at two time points. Chemotherapeutic treatments induced distinct changes in ncRNA expression in TNBC cell lines and the BT­474 cell line in intra­ and extracellular compartments. Serum and urine­based ncRNA expression analysis was able to discriminate between patients with TNBC and controls. Time point comparisons in the urine samples of patients with TNBC revealed a general rise in the level of ncRNA. Serum data suggested a potential association between piR­36743, miR­17, ­19b and ­30b expression levels and an NACT­driven complete clinical response. The present study highlighted the potential of ncRNAs as liquid biopsy­based biomarkers in TNBC chemotherapy treatment. The ncRNAs tested in the present study have been previously investigated for their involvement in BC or TNBC chemotherapy responses; however, these previous studies were restricted to patient tissue or in vitro models. The data from the present study offer novel insight into ncRNA expression in liquid samples from patients with TNBC, and the study serves as an initial step in the evaluation of ncRNAs as diagnostic biomarkers in the monitoring of TNBC therapy.

Addition of IMP3 to L1CAM for discrimination between low- and high-grade endometrial carcinomas: a European Network for Individualised Treatment of Endometrial Cancer collaboration study.

Discrimination between low- and high-grade endometrial carcinomas (ECs) is clinically relevant but can be challenging for pathologists, with moderate interobserver agreement. Insulin-like growth factor-II mRNA-binding protein 3 (IMP3) is an oncofoetal protein that is associated with nonendometrioid endometrial carcinomas but has been limited studied in endometrioid carcinomas. The aim of this study is to investigate the diagnostic and prognostic value of IMP3 in the discrimination between low- and high-grade ECs and its added value to L1CAM. IMP3 and L1CAM expression was assessed in tumors from 378 patients treated for EC at 1 of 9 participating European Network for Individualised Treatment of Endometrial Cancer centers. IMP3 was expressed in 24.6% of the tumors. In general, IMP3 was more homogeneously expressed than L1CAM. IMP3 expression was significantly associated with advanced stage, nonendometrioid histology, grade 3 tumors, deep myometrial invasion, lymphovascular space invasion, distant recurrences, overall mortality, and disease-related mortality. Simultaneous absence of IMP3 and L1CAM expression showed the highest accuracy for identifying low-grade carcinomas (area under the curve 0.766), whereas simultaneous expression of IMP3 and L1CAM was strongly associated with high-grade carcinomas (odds ratio 19.7; 95% confidence interval 9.2-42.2). Even within endometrioid carcinomas, this combination remained superior to IMP3 and L1CAM alone (odds ratio 8.6; 95% confidence interval 3.4-21.9). In conclusion, IMP3 has good diagnostic value and together with L1CAM represents the optimal combination of diagnostic markers for discrimination between low- and high-grade ECs compared to IMP3 and L1CAM alone. Because of the homogenous expression of IMP3, this marker might be valuable in preoperative biopsies when compared to the more patchy L1CAM expression.

Poor outcome in hypoxic endometrial carcinoma is related to vascular density.

BACKGROUND: Identification of endometrial carcinoma (EC) patients at high risk of recurrence is lacking. In this study, the prognostic role of hypoxia and angiogenesis was investigated in EC patients. METHODS: Tumour slides from EC patients were stained by immunofluorescence for carbonic anhydrase IX (CAIX) as hypoxic marker and CD34 for assessment of microvessel density (MVD). CAIX expression was determined in epithelial tumour cells, with a cut-off of 1%. MVD was assessed according to the Weidner method. Correlations with disease-specific survival (DSS), disease-free survival (DFS) and distant disease-free survival (DDFS) were calculated using Kaplan-Meier curves and Cox regression analysis. RESULTS: Sixty-three (16.4%) of 385 ECs showed positive CAIX expression with high vascular density. These ECs had a reduced DSS compared to tumours with either hypoxia or high vascular density (log-rank p = 0.002). Multivariable analysis showed that hypoxic tumours with high vascular density had a reduced DSS (hazard ratio [HR] 3.71, p = 0.002), DDFS (HR 2.68, p = 0.009) and a trend for reduced DFS (HR 1.87, p = 0.054). CONCLUSIONS: This study has shown that adverse outcome in hypoxic ECs is seen in the presence of high vascular density, suggesting an important role of angiogenesis in the metastatic process of hypoxic EC. Differential adjuvant treatment might be indicated for these patients.