Baseline CTC Count as a Predictor of Long-Term Outcomes in High-Risk Prostate Cancer.

The aim of the present study was to verify whether the baseline circulating tumor cell (CTC) count might serve as a predictor of overall survival (OS) and metastasis-free survival (MFS) in patients with high-risk prostate cancer (PCa) during a follow-up period of at least 5 years. CTCs were enumerated using three different assay formats in 104 patients: the CellSearch® system, EPISPOT assay and GILUPI CellCollector. A total of 57 (55%) patients survived until the end of the follow-up period, with a 5 year OS of 66% (95% CI: 56-74%). The analysis of univariate Cox proportional hazard models identified a baseline CTC count ≥ 1, which was determined with the CellSearch® system, a Gleason sum ≥ 8, cT ≥ 2c and metastases at initial diagnosis as significant predictors of a worse OS in the entire cohort. The CTC count ≥ 1 was also the only significant predictor of a worse OS in a subset of 85 patients who presented with localized PCa at the baseline. The baseline CTC number did not affect the MFS. In conclusion, the baseline CTC count can be considered a determinant of survival in high-risk PCa and also in patients with a localized disease. However, determining the prognostic value of the CTC count in patients with localized PCa would optimally require longitudinal monitoring of this parameter.

Circulating Melanoma Cell Numbers Correlate with TIGIT-Positive Cytotoxic T Cell Counts in Advanced-Stage Melanoma Patients.

Despite the rising public awareness of the risk factors and the possible prevention of melanoma development, it remains challenging in terms of diagnosis and treatment. To improve the clinical situation of patients, it would be especially beneficial to develop prognostic methods for the effective and continuous assessment of the disease course. The solution could lie in the selection of effective biomarkers derived from the tumor microenvironment, increasing the effectiveness of melanoma prognoses and monitoring. Hence, in this study, we evaluated the number of circulating melanoma cells (CMCs) in representative blood samples of melanoma patients vs. healthy controls, as well as the proportion of particular cytotoxic T cells in the total lymphocyte and leukocyte population as a reflection of immune resistance. The results were correlated with the clinical parameters of the patients to examine the potential value of CMC quantification and lymphoid cell phenotyping in melanoma diagnostics, prognostics, and treatment outcome monitoring. The CMC numbers were significantly higher in melanoma patients than in healthy controls. However, an analysis of the correlations between the baseline CMC counts and the clinical parameters found no significant results. In turn, we found significant differences between the groups in the percentage of various profiles of CD8+ cytotoxic T lymphocytes characterized by TIGIT and TIM-3 differential expression. Importantly, the CMC number correlated with CD8+TIGIT+ and CD8+TIGIT+TIM-3- cytotoxic T cell counts in the melanoma patient group. Considering the above, the combination of CMCs and the immunological status of the patient, as defined by the prevalence of selected immune cell types, seems to be a promising approach in melanoma diagnostics and prognostics.

MicroRNA in lung cancer-a novel potential way for early diagnosis and therapy.

Lung cancer is the most common cause of cancer-related deaths in the world. One of the reasons of poor prognosis and high mortality of lung cancer patients is the diagnosis of the disease in its advanced stage. Despite innovative diagnostic methods and multiple completed and ongoing clinical trials aiming at therapy improvement, no significant increase in patients' long-term survival has been noted over last decades. Patients would certainly benefit from early detection of lung cancer. Therefore, it is crucial to find new biomarkers that can help predict outcomes and tumor responses in order to maximize therapy effectiveness and avoid over- or under-treating patients with lung cancer. Nowadays, scientists' attention is mainly dedicated to so-called liquid biopsy, which is fully non-invasive and easily available method based on simple blood draw. Among common liquid biopsy elements, circulating tumor nucleic acids are worth mentioning. Epigenetic biomarkers, particularly miRNA expression, have several distinct features that make them promising prognostic markers. In this review, we described miRNA's involvement in tumorigenesis and present it as a predictor of cancer development and progression, potential indicator of treatment efficacy, and most importantly promising therapeutic target.

Liquid Biopsy Analysis as a Tool for TKI-Based Treatment in Non-Small Cell Lung Cancer.

The treatment of non-small cell lung cancer (NSCLC) has recently evolved with the introduction of targeted therapy based on the use of tyrosine kinase inhibitors (TKIs) in patients with certain gene alterations, including EGFR, ALK, ROS1, BRAF, and MET genes. Molecular targeted therapy based on TKIs has improved clinical outcomes in a large number of NSCLC patients with advanced disease, enabling significantly longer progression-free survival (PFS). Liquid biopsy is an increasingly popular diagnostic tool for treating TKI-based NSCLC. The studies presented in this article show that detection and analysis based on liquid biopsy elements such as circulating tumor cells (CTCs), cell-free DNA (cfDNA), exosomes, and/or tumor-educated platelets (TEPs) can contribute to the appropriate selection and monitoring of targeted therapy in NSCLC patients as complementary to invasive tissue biopsy. The detection of these elements, combined with their molecular analysis (using, e.g., digital PCR (dPCR), next generation sequencing (NGS), shallow whole genome sequencing (sWGS)), enables the detection of mutations, which are required for the TKI treatment. Despite such promising results obtained by many research teams, it is still necessary to carry out prospective studies on a larger group of patients in order to validate these methods before their application in clinical practice.

Liquid Biopsy in Melanoma: Significance in Diagnostics, Prediction and Treatment Monitoring.

Liquid biopsy is a common term referring to circulating tumor cells and other biomarkers, such as circulating tumor DNA (ctDNA) or extracellular vesicles. Liquid biopsy presents a range of clinical advantages, such as the low invasiveness of the blood sample collection and continuous control of the tumor progression. In addition, this approach enables the mechanisms of drug resistance to be determined in various methods of cancer treatment, including immunotherapy. However, in the case of melanoma, the application of liquid biopsy in patient stratification and therapy needs further investigation. This review attempts to collect all of the relevant and recent information about circulating melanoma cells (CMCs) related to the context of malignant melanoma and immunotherapy. Furthermore, the biology of liquid biopsy analytes, including CMCs, ctDNA, mRNA and exosomes, as well as techniques for their detection and isolation, are also described. The available data support the notion that thoughtful selection of biomarkers and technologies for their detection can contribute to the development of precision medicine by increasing the efficacy of cancer diagnostics and treatment.

Clinical Relevance of Circulating Tumor Cells in Prostate Cancer Management.

Given the low specificity of the routinely used biomarker prostate-specific antigen, circulating tumor cell (CTC) enumeration seems to be particularly useful in the monitoring of prostate cancer. In this review, we focused on a few aspects of CTC enumeration in prostate malignancies: prognostic value in metastatic and non-metastatic tumors, role in the monitoring of treatment outcomes, use as a surrogate marker for survival, and other applications, mostly for research purposes. CTC enumeration, without a doubt, offers an attractive perspective in the management of prostate cancer. However, the vast majority of available data about the role of CTC in this malignancy originate from randomized studies of anticancer agents and do not necessarily translate into real-world clinical practice. Further, most studies on the application of CTC in prostate cancer patients were limited to advanced stages of this malignancy. Meanwhile, the role of CTC in the early stages of prostate cancer, in which some patients may present with occult disseminated disease, is still relatively poorly understood, and should thus be studied extensively. Other obstacles in the widespread application of CTC enumeration in routine clinical practice include considerable discrepancies in the number of cells determined with various commercially available systems.

[Corrigendum] Novel markers of human ovarian granulosa cell differentiation toward osteoblast lineage: A microarray approach.

Following the publication of this paper, the authors have requested that, on p. 4412 of the above article in the Funding section of the Declarations, the acknowledgement to one of the funding sources should be removed from the paper; essentially, the reference to grant no. 2018/31/B/NZ5/02475, formulated by the Polish National Science Centre (grant providing institution), should be removed from the paper. Therefore, the revised version of the Funding section paragraph should read as follows: Funding: The present study was supported by a grant from Poznan University of Medical Sciences (grant no. 502­14­02227367­10694). The authors confirm that there are no further errors in the study, and all the authors agree to this correction. The authors are grateful to the Editor of Molecular Medicine Reports for granting them this opportunity to publish a Corrigendum, and apologize for any inconvenience caused. [the original article was published in Molecular Medicine Reports 20: 4403-4414, 2019, DOI: 10.3892/mmr.2019.10709].

New Gene Markers Expressed in Porcine Oviductal Epithelial Cells Cultured Primary In Vitro Are Involved in Ontological Groups Representing Physiological Processes of Porcine Oocytes.

Changes that occur within oviducts after fertilization are dependent on post-ovulation events, including oocyte-oviduct interactions. Although general processes are well-defined, the molecular basis are poorly understood. Recently, new marker genes involved in 'cell development', 'cell growth', 'cell differentiation' and 'cell maturation' processes have been identified in porcine oocytes. The aim of the study was to assess the expression profile of genes in primary in vitro cultured oviductal epithelial cells (OECs), clustered in Gene Ontology groups which enveloped markers also identified in porcine oocytes. OECs (from 45 gilts) were surgically removed and cultured in vitro for ≤ 30 days, and then subjected to molecular analyses. The transcriptomic and proteomic profiles of cells cultured during 7, 15 and 30 days were investigated. Additionally, morphological/histochemical analyzes were performed. The results of genes expression profiles were validated after using RT-qPCR. The results showed a significant upregulation of UNC45B, NOX4, VLDLR, ITGB3, FMOD, SGCE, COL1A2, LOX, LIPG, THY1 and downregulation of SERPINB2, CD274, TXNIP, CELA1, DDX60, CRABP2, SLC5A1, IDO1, ANPEP, FST. Detailed knowledge of the molecular pathways occurring in the OECs and the gametes that contact them may contribute both to developments of basic science of physiology, and new possibilities in advanced biotechnology of assisted reproduction.

Human Cumulus Cells in Long-Term In Vitro Culture Reflect Differential Expression Profile of Genes Responsible for Planned Cell Death and Aging-A Study of New Molecular Markers.

In the ovarian follicle, maturation of the oocyte increases in the presence of somatic cells called cumulus cells (CCs). These cells form a direct barrier between the oocyte and external environment. Thanks to bidirectional communication, they have a direct impact on the oocyte, its quality and development potential. Understanding the genetic profile of CCs appears to be important in elucidating the physiology of oocytes. Long-term in vitro culture of CCs collected from patients undergoing controlled ovarian stimulation during in vitro fertilization procedure was conducted. Using microarray expression analysis, transcript levels were assessed on day 1, 7, 15, and 30 of culture. Apoptosis and aging of CCs strictly influence oocyte quality and subsequently the outcome of assisted reproductive technologies (ART). Thus, particular attention was paid to the analysis of genes involved in programmed cell death, aging, and apoptosis. Due to the detailed level of expression analysis of each of the 133 analyzed genes, three groups were selected: first with significantly decreased expression during the culture; second with the statistically lowest increase in expression; and third with the highest significant increase in expression. COL3A1, SFRP4, CTGF, HTR2B, VCAM1, TNFRSF11B genes, belonging to the third group, were identified as potential carriers of information on oocyte quality.

Circulating Tumor Cells as a Marker of Disseminated Disease in Patients with Newly Diagnosed High-Risk Prostate Cancer.

The aim of this study was to investigate whether the enumeration of circulating tumor cells (CTCs) in blood can differentiate between true localized and metastatic prostate cancer. A cross-sectional study of 104 prostate cancer patients with newly diagnosed high-risk prostate cancer was conducted. In total, 19 patients presented metastatic disease and 85 were diagnosed with localized disease. Analyses included intergroup comparison of CTC counts, determined using the CellSearch® system, EPISPOT assay and GILUPI CellCollector®, and ROC analysis verifying the accuracy of CTC count as a maker of disseminated prostate cancer. The vast majority (94.7%) of patients with advanced-stage cancer tested positively for CTCs in at least one of the assays. However, significantly higher CTC counts were determined with the CellSearch® system compared to EPISPOT assay and GILUPI CellCollector®. Identification of ≥4 CTCs with the CellSearch® system was the most accurate predictor of metastatic disease (sensitivity 0.500; specificity 0.900; AUC (95% CI) 0.760 (0.613-0.908). Furthermore, we tried to create a model to enhance the specificity and sensitivity of metastatic prediction with CTC counts by incorporating patient's clinical data, including PSA serum levels, Gleason score and clinical stage. The composite biomarker panel achieved the following performance: sensitivity, 0.611; specificity, 0.971; AUC (95% CI), 0.901 (0.810-0.993). Thus, although the sensitivity of CTC detection needs to be further increased, our findings suggest that high CTC counts might contribute to the identification of high-risk prostate cancer patients with occult metastases at the time of diagnosis.

Novel markers of human ovarian granulosa cell differentiation toward osteoblast lineage: A microarray approach.

Under physiological conditions, human ovarian granulosa cells (GCs), are responsible for a number of processes associated with folliculogenesis and oogenesis. The primary functions of GCs in the individual phases of follicle growth are: Hormone production in response to follicle stimulating hormone (FSH), induction of ovarian follicle atresia through specific molecular markers and production of nexus cellular connections for communication with the oocyte. In recent years, interest in obtaining stem cells from particular tissues, including the ovary, has increased. Special attention has been paid to the novel properties of GCs during long­term in vitro culture. It has been demonstrated that the usually recycled material in the form of follicular fluid can be a source of cells with stem­like properties. The study group consisted of patients enrolled in the in vitro fertilization procedure. Total RNA was isolated from GCs at 4 time points (after 1, 7, 15 and 30 days of culture) and was used for microarray expression analysis (Affymetrix® Human HgU 219 Array). The expression of 22,480 transcripts was examined. The selection of significantly altered genes was based on a P­value <0.05 and expression higher than two­fold. The leucine rich repeat containing 17, collagen type I α1 chain, bone morphogenetic protein 4, twist family bHLH transcription factor 1, insulin like growth factor binding protein 5, GLI family zinc finger 2 and collagen triple helix repeat containing genes exhibited the highest changes in expression. Reverse­transcription­quantitative PCR was performed to validate the results obtained in the analysis of expression microarrays. The direction of expression changes was validated in the majority of cases. The presented results indicated that GCs have the potential of cells that can differentiate towards osteoblasts in long­term in vitro culture conditions. Increased expression of genes associated with the osteogenesis process suggests a potential for uninduced change of GC properties towards the osteoblast phenotype. The present study, therefore, suggests that GCs may become an excellent starting material in obtaining stable osteoblast cultures. GCs differentiated towards osteoblasts may be used in regenerative and reconstructive medicine in the future.

"Biological Adhesion" is a Significantly Regulated Molecular Process during Long-Term Primary In Vitro Culture of Oviductal Epithelial Cells (Oecs): A Transcriptomic and Proteomic Study.

Oviductal epithelial cells (OECs) actively produce stimulating and protecting factors, favoring survival and viability of gametes and early embryos. The oviduct participates in the initial reproductive events, which strongly depends on adhesion. The analysis of differential gene expression in OECs, during long-term in vitro culture, enables recognition of new molecular markers regulating several processes, including "biological adhesion". Porcine oviducts were stained with hematoxylin and eosin, as well as with antibodies against epithelial markers. Then, OECs were long-term in vitro cultured and after 24 h, 7, 15, and 30 days of culture were subjected to transcriptomic and proteomic assays. Microarrays were employed to evaluate gene expression, with Matrix-assisted laser desorption/ionization-time of light (MALDI-TOF) mass spectrometry applied to determine the proteome. The results revealed proper morphology of the oviducts and typical epithelial structure of OECs during the culture. From the set of differentially expressed genes (DEGs), we have selected the 130 that encoded proteins detected by MALDI-TOF MS analysis. From this gene pool, 18 significantly enriched gene ontology biological processes (GO BP) terms were extracted. Among them we focused on genes belonging to "biological adhesion" GO BP. It is suggested that increased expression of studied genes can be attributed to the process of intensive secretion of substances that exhibit favorable influence on oviductal environment, which prime gametes adhesion and viability, fertilization, and early embryo journey.

Analysis of Circulating Tumor Cells in Patients with Non-Metastatic High-Risk Prostate Cancer before and after Radiotherapy Using Three Different Enumeration Assays.

The characterization of circulating tumor cells (CTCs) can lead to a promising strategy for monitoring residual or relapsing prostate cancer (PCa) after local therapy. The aim of this study was to compare three innovative technologies for CTC enumeration in 131 high-risk patients with PCa, before and after radiotherapy, combined with androgen deprivation. The CTC number was tested using the FDA-cleared CellSearch® system, the dual fluoro-EPISPOT assay that only detects functional CTCs, and the in vivo CellCollector® technology. The highest percentage of CTC-positive patients was detected with the CellCollector® (48%) and dual fluoro-EPISPOT (42%) assays, while the CellSearch® system presented the lowest rate (14%). Although the concordance among methods was only 23%, the cumulative positivity rate was 79%. A matched-pair analysis of the samples before, and after, treatment suggested a trend toward a decrease in CTC count after treatment with all methods. CTC tended to be positivity correlated with age for the fluoro-EPISPOT assay and with PSA level from the data of three assays. Combining different CTC assays improved CTC detection rates in patients with non-metastatic high-risk PCa before and after treatment. Our findings do not support the hypothesis that radiotherapy leads to cancer cell release in the circulation.

Transcriptomic Pattern of Genes Regulating Protein Response and Status of Mitochondrial Activity Are Related to Oocyte Maturational Competence-A Transcriptomic Study.

This paper aims to identify and describe new genetic markers involved in the processes of protein expression and modification reflected in the change of mitochondrial activity before and after in vitro maturation of the oocyte. Porcine oocytes collected from the ovaries of slaughtered landrace gilts were subjected to the process of in vitro maturation. Transcriptomic changes in the expression profile of oocyte genes involved in response to hypoxia, the transmembrane protein receptor serine threonine kinase signaling pathway, the "transforming growth factor ß receptor signaling pathway", "response to protein stimulus", and "response to organic substance" were investigated using microarrays. The expression values of these genes in oocytes was analyzed before (immature) and after (mature) in vitro maturation, with significant differences found. All the significantly altered genes showed downregulation after the maturation process. The most changed genes from these gene ontologies, FOS, ID2, VEGFA, BTG2, CYR61, ESR1, AR, TACR3, CCND2, CHRDL1, were chosen to be further validated, described and related to the literature. Additionally, the mitochondrial activity of the analyzed oocytes was measured using specific dyes. We found that the mitochondrial activity was higher before the maturation process. The analysis of these results and the available literature provides a novel insight on the processes that occur during in vitro oocyte maturation. While this knowledge may prove to be useful in further research of the procedures commonly associated with in vitro fertilization procedures, it serves mostly as a basic reference for further proteomic, in vivo, and clinical studies that are necessary to translate it into practical applications.

The Unique Mechanisms of Cellular Proliferation, Migration and Apoptosis are Regulated through Oocyte Maturational Development-A Complete Transcriptomic and Histochemical Study.

The growth and development of oocyte affect the functional activities of the surrounding somatic cells. These cells are regulated by various types of hormones, proteins, metabolites, and regulatory molecules through gap communication, ultimately leading to the development and maturation of oocytes. The close association between somatic cells and oocytes, which together form the cumulus-oocyte complexes (COCs), and their bi-directional communication are crucial for the acquisition of developmental competences by the oocyte. In this study, oocytes were extracted from the ovaries obtained from crossbred landrace gilts and subjected to in vitro maturation. RNA isolated from those oocytes was used for the subsequent microarray analysis. The data obtained shows, for the first time, variable levels of gene expression (fold changes higher than |2| and adjusted p-value < 0.05) belonging to four ontological groups: regulation of cell proliferation (GO:0042127), regulation of cell migration (GO:0030334), and regulation of programmed cell death (GO:0043067) that can be used together as proliferation, migration or apoptosis markers. We have identified several genes of porcine oocytes (ID2, VEGFA, BTG2, ESR1, CCND2, EDNRA, ANGPTL4, TGFBR3, GJA1, LAMA2, KIT, TPM1, VCP, GRID2, MEF2C, RPS3A, PLD1, BTG3, CD47, MITF), whose expression after in vitro maturation (IVM) is downregulated with different degrees. Our results may be helpful in further elucidating the molecular basis and functional significance of a number of gene markers associated with the processes of migration, proliferation and angiogenesis occurring in COCs.