EGFR and PI3K Signalling Pathways as Promising Targets on Circulating Tumour Cells from Patients with Metastatic Gastric Adenocarcinoma.

The prognosis for metastatic gastric adenocarcinoma (mGAC) remains poor. Gene alterations in receptor tyrosine kinases (RTKs) such as epidermal growth factor receptor (EGFR) and their downstream effectors including catalytic subunit alpha of the phosphatidylinositol 3-kinase (PIK3CA) are common in mGAC. Targeted RTK and phosphatidylinositol-3-kinase (PI3K) treatments have demonstrated clinical benefits in other solid tumours and are key potential targets for clinical development against mGAC given the presence of recurrent alterations in these pathways. Furthermore, combination RTK/PI3K treatments may overcome compensatory mechanisms that arise using monotherapies, leading to improved patient outcomes. Herein, we investigated RTK/PI3K single and combination drug responses against our unique human mGAC-derived PIK3CA gain-of-function mutant, human epidermal growth factor receptor 2 (HER2)-negative, EGFR-expressing circulating tumour cell line, UWG02CTC, under two- and three-dimensional culture conditions to model different stages of metastasis. UWG02CTCs were highly responsive to the PI3K p110α-subunit targeted drugs PIK-75 (IC50 = 37.0 ± 11.1 nM) or alpelisib (7.05 ± 3.7 µM). Drug sensitivities were significantly increased in 3D conditions. Compensatory MAPK/ERK pathway upregulation by PI3K/Akt suppression was overcome by combination treatment with the EGFR inhibitor gefitinib, which was strongly synergistic. PIK-75 plus gefitinib significantly impaired UWG02CTC invasion in an organotypic assay. In conclusion, UWG02CTCs are a powerful ex vivo mGAC drug responsiveness model revealing EGFR/PI3K-targeted drugs as a promising combination treatment option for HER2-negative, RAS wild-type mGAC patients.

Single-Circulating Tumor Cell Whole Genome Amplification to Unravel Cancer Heterogeneity and Actionable Biomarkers.

The field of single-cell analysis has advanced rapidly in the last decade and is providing new insights into the characterization of intercellular genetic heterogeneity and complexity, especially in human cancer. In this regard, analyzing single circulating tumor cells (CTCs) is becoming particularly attractive due to the easy access to CTCs from simple blood samples called "liquid biopsies". Analysis of multiple single CTCs has the potential to allow the identification and characterization of cancer heterogeneity to guide best therapy and predict therapeutic response. However, single-CTC analysis is restricted by the low amounts of DNA in a single cell genome. Whole genome amplification (WGA) techniques have emerged as a key step, enabling single-cell downstream molecular analysis. Here, we provide an overview of recent advances in WGA and their applications in the genetic analysis of single CTCs, along with prospective views towards clinical applications. First, we focus on the technical challenges of isolating and recovering single CTCs and then explore different WGA methodologies and recent developments which have been utilized to amplify single cell genomes for further downstream analysis. Lastly, we list a portfolio of CTC studies which employ WGA and single-cell analysis for genetic heterogeneity and biomarker detection.

Molecular Biomarkers in Glioblastoma: A Systematic Review and Meta-Analysis.

Background: Glioblastoma (GBM) is a highly aggressive cancer with poor prognosis that needs better treatment modalities. Moreover, there is a lack of reliable biomarkers to predict the response and outcome of current or newly designed therapies. While several molecular markers have been proposed as potential biomarkers for GBM, their uptake into clinical settings is slow and impeded by marker heterogeneity. Detailed assessment of prognostic and predictive value for biomarkers in well-defined clinical trial settings, if available, is scattered throughout the literature. Here we conducted a systematic review and meta-analysis to evaluate the prognostic and predictive significance of clinically relevant molecular biomarkers in GBM patients. Material and methods: A comprehensive literature search was conducted to retrieve publications from 3 databases (Pubmed, Cochrane and Embase) from January 2010 to December 2021, using specific terms. The combined hazard ratios (HR) and confidence intervals (95% CI) were used to evaluate the association of biomarkers with overall survival (OS) in GBM patients. Results: Twenty-six out of 1831 screened articles were included in this review. Nineteen articles were included in the meta-analyses, and 7 articles were quantitatively summarised. Fourteen studies with 1231 GBM patients showed a significant association of MGMT methylation with better OS with the pooled HR of 1.66 (95% CI 1.32−2.09, p < 0.0001, random effect). Five studies including 541 GBM patients analysed for the prognostic significance of IDH1 mutation showed significantly better OS in patients with IDH1 mutation with a pooled HR of 2.37 (95% CI 1.81−3.12; p < 0.00001]. Meta-analysis performed on 5 studies including 575 GBM patients presenting with either amplification or high expression of EGFR gene did not reveal any prognostic significance with a pooled HR of 1.31 (95% CI 0.96−1.79; p = 0.08). Conclusions: MGMT promoter methylation and IDH1 mutation are significantly associated with better OS in GBM patients. No significant associations were found between EGFR amplification or overexpression with OS.

Distinguishing rosacea from sensitive skin by reflectance confocal microscopy.

BACKGROUND: The updated standard classification and pathophysiology of rosacea have provided clear and meaningful evaluation parameters; however, differentiating rosacea from sensitive skin (SS) remained an obstacle for dermatologists around the world, especially in China. Herein, we aimed to find a better characteristic to distinguish rosacea from SS by using reflectance confocal microscopy (RCM). METHOD: Forty rosacea patients and 143 healthy subjects were recruited in this study. Firstly, a SS questionnaire and a lactic acid sting test were conducted among healthy subjects. Next, two major groups were divided out, including a SS group (40 subjects) and a normal skin control group (NS, 60 subjects). The cutaneous structures of face and fossa cubitalia were imaged by RCM. RESULTS: We found that more parakeratosis, honeycomb pattern, spongiform edema, and dermal papillae (P < .05) in rosacea patients than that of the NS group, whereas there were no significant differences, were found in rosacea patients and the SS group. Strikingly, we found that rosacea patients have a larger depth of honeycomb pattern than that of SS subjects (P < .05). But, the epidermal thickness of rosacea did not differ from that of SS groups. There was also no significant difference of epidermal thickness and honeycomb structure depth between rosacea patients and NS group. CONCLUSION: From the RCM images of parakeratosis, honeycomb pattern, spongiform edema, and dermal papillae, we found that RCM might be a faithful tool to distinguish rosacea from NS group. The depth of honeycomb structure of SS was more superficial than rosacea patients, whereas no significant difference between rosacea patients and NS group. RCM may provide a new method for evaluating the development of rosacea although it failed to distinguish rosacea and SS effectively.

Rosacea is associated with conjoined interactions between physical barrier of the skin and microorganisms: A pilot study.

BACKGROUND: Rosacea is a common condition characterized by transient or persistent central facial erythema, and often papules and pustules. Currently, the role of bacterium in the development and progression of rosacea remains controversial. This study aimed to investigate the difference in the physiological conditions and microorganisms between the lesional and non-lesional areas of papulopustular rosacea. METHODS: Twenty-five French patients with papulopustular rosacea were enrolled in this pilot study. Each patient was subjected to clinical assessment, and the skin barrier function was tested in lesional and non-lesional areas. In addition, samples from the lesional and non-lesional areas were collected for bacterial culturing. RESULTS: Of all subjects included in the study, a lower skin conductivity was measured in lesional areas than in non-lesional areas (43.5 ± 12.4 vs. 57.2 ± 11.6 U, P < .05), and a higher transepidermal water loss (TEWL) value was found in lesional areas than in non-lesional areas (17.2 ± 5.9 vs. 14.2 ± 4.1 g/(m2  h), P < .05). We found a lower TEWL in lesions in rosacea patients with bacterial dysbiosis than in those with bacterial balance (P < .05). In addition, there were significant differences in the skin conductivity and TEWL between lesional and non-lesional areas in patients with bacterial dysbiosis (P < .001), and no significant differences were seen in patients with bacterial balance (P < .05). CONCLUSION: The results of the present study demonstrate that the physiological features of rosacea are closely associated with the interactions between the host and the microorganisms.

Differential gene expression for carotenoid biosynthesis in a green alga Ulva prolifera based on transcriptome analysis.

BACKGROUND: Carotenoids are widely distributed in plants and algae, and their biosynthesis has attracted widespread interest. Carotenoid-related research has mostly focused on model species, and there is a lack of data on the carotenoid biosynthetic pathway in U. prolifera that is the main species leading to green tide, a harmful plague of floating green algae. RESULTS: The carotenoid content of U. prolifera samples, that is the main species leading to green tide, a harmful plague of floating green algae at different temperatures revealed that its terpenoid was highest in the samples subjected to high temperature at 28 °C (H), followed by the samples subjected to low temperature at 12 °C (L). Its terpenoid was lowest in the samples subjected to medium temperature at 20 °C (M). We conducted transcriptome sequencing (148.5 million raw reads and 49,676 unigenes in total) of samples that were subjected to different temperatures to study the carotenoid biosynthesis of U. prolifera. There were 1125-3164 significant differentially expressed genes between L, M and H incubation temperatures, of which 11-672 genes were upregulated and 453-3102 genes were downregulated. A total of 3164 genes were significantly differentially expressed between H and M, of which 62 genes were upregulated and 3102 genes were downregulated. A total of 2669 significant differentially expressed genes were observed between L and H, of which 11 genes were upregulated and 2658 genes were downregulated. A total of 13 genes were identified to be involved in carotenoid biosynthesis in U. prolifera, and the expression levels of the majority were highest at H and lowest at M of incubation temperature. Both the carotenoid concentrations and the expression of the analysed genes were lowest in the normal temperature group, while low temperature and high temperature seemed to activate the biosynthesis of carotenoids in U. prolifera. CONCLUSIONS: In this study, transcriptome sequencing provided critical information for understanding the accumulation of carotenoids and will serve as an important reference for the study of other metabolic pathways in U. prolifera.

CTC-mRNA (AR-V7) Analysis from Blood Samples-Impact of Blood Collection Tube and Storage Time.

Circulating tumour cells (CTCs) are an emerging resource for monitoring cancer biomarkers. New technologies for CTC isolation and biomarker detection are increasingly sensitive, however, the ideal blood storage conditions to preserve CTC-specific mRNA biomarkers remains undetermined. Here we tested the preservation of tumour cells and CTC-mRNA over time in common anticoagulant ethylene-diamine-tetra-acetic acid (EDTA) and acid citrate dextrose solution B (Citrate) blood tubes compared to preservative-containing blood tubes. Blood samples spiked with prostate cancer cells were processed after 0, 24, 30, and 48 h storage at room temperature. The tumour cell isolation efficiency and the mRNA levels of the prostate cancer biomarkers androgen receptor variant 7 (AR-V7) and total AR, as well as epithelial cell adhesion molecule (EpCAM) were measured. Spiked cells were recovered across all storage tube types and times. Surprisingly, tumour mRNA biomarkers were readily detectable after 48 h storage in EDTA and Citrate tubes, but not in preservative-containing tubes. Notably, AR-V7 expression was detected in prostate cancer patient blood samples after 48 h storage in EDTA tubes at room temperature. This important finding presents opportunities for measuring AR-V7 expression from clinical trial patient samples processed within 48 h-a much more feasible timeframe compared to previous recommendations.

Fascin 1 is transiently expressed in mouse melanoblasts during development and promotes migration and proliferation.

Fascins, a family of actin-bundling proteins, are expressed in a spatially and temporally restricted manner during development and often in cancer. Fascin 1 has a clear role in cell migration in vitro, but its role in vivo in mammals is not well understood. Here, we investigate the role of fascin 1 in the melanocyte lineage and in melanoma cells. Fascin 1 knockout causes hypopigmentation in adult mice owing to migration and cell cycle progression defects in melanoblasts, the melanocyte precursor cell. Study of live embryo skin explants reveals that E14.5 fascin 1-null melanoblasts migrate slower, and generate fewer and thinner pseudopods. By contrast, fascin 1 expression drives faster migration and lamellipodia protrusion in melanocytes in vitro. In addition, fascin 1 depletion retards melanoblast proliferation in vivo and melanoma cell growth in vitro. These data indicate that fascin 1 not only promotes cell migration in mouse melanocytes but it also has a role in growth and cell cycle progression.

Droplet Digital PCR Based Androgen Receptor Variant 7 (AR-V7) Detection from Prostate Cancer Patient Blood Biopsies.

Androgen receptor splice variant V7 (AR-V7) was recently identified as a valuable predictive biomarker in metastatic castrate-resistant prostate cancer. Here, we report a new, sensitive and accurate screen for AR-V7 mRNA expression directly from circulating tumor cells (CTCs): We combined EpCAM-based immunomagnetic CTC isolation using the IsoFlux microfluidic platform with droplet digital polymerase chain reaction (ddPCR) to analyze total AR and AR-V7 expression from prostate cancer patients CTCs. We demonstrate that AR-V7 is reliably detectable in enriched CTC samples with as little as five CTCs, even considering tumor heterogeneity, and confirm detection of AR-V7 in CTC samples from advanced prostate cancer (PCa) patients with AR-V7 detection limited to castrate resistant disease status in our sample set. Sensitive molecular analyses of circulating tumor cells (CTCs) or circulating tumor nucleic acids present exciting strategies to detect biomarkers, such as AR-V7 from non-invasive blood samples, so-called blood biopsies.

Fascin1 in carcinomas: Its regulation and prognostic value.

Previous cell biological studies demonstrate that the actin bundling protein fascin1 regulates cell motility, migration and invasion. Human studies demonstrate that fascin1 is upregulated in many epithelial cancers. This review gives a brief overview of the role of fascin1 in cell migration and invasion, but focuses mainly on the regulation and clinical relevance of fascin1 in epithelial cancers. Here, we propose fascin1 as a potent prognostic biomarker for breast, colorectal, esophageal cancers and head and neck squamous cell carcinomas. Fascin1 may also be an attractive drug target against these carcinomas in the future, but more studies are needed.

Fascin is regulated by slug, promotes progression of pancreatic cancer in mice, and is associated with patient outcomes.

BACKGROUND & AIMS: Pancreatic ductal adenocarcinoma (PDAC) is often lethal because it is highly invasive and metastasizes rapidly. The actin-bundling protein fascin has been identified as a biomarker of invasive and advanced PDAC and regulates cell migration and invasion in vitro. We investigated fascin expression and its role in PDAC progression in mice. METHODS: We used KRas(G12D) p53(R172H) Pdx1-Cre (KPC) mice to investigate the effects of fascin deficiency on development of pancreatic intraepithelial neoplasia (PanIn), PDAC, and metastasis. We measured levels of fascin in PDAC cell lines and 122 human resected PDAC samples, along with normal ductal and acinar tissues; we associated levels with patient outcomes. RESULTS: Pancreatic ducts and acini from control mice and early-stage PanINs from KPC mice were negative for fascin, but approximately 6% of PanIN3 and 100% of PDAC expressed fascin. Fascin-deficient KRas(G12D) p53(R172H) Pdx1-Cre mice had longer survival times, delayed onset of PDAC, and a lower PDAC tumor burdens than KPC mice; loss of fascin did not affect invasion of PDAC into bowel or peritoneum in mice. Levels of slug and fascin correlated in PDAC cells; slug was found to regulate transcription of Fascin along with the epithelial-mesenchymal transition. In PDAC cell lines and cells from mice, fascin concentrated in filopodia and was required for their assembly and turnover. Fascin promoted intercalation of filopodia into mesothelial cell layers and cell invasion. Nearly all human PDAC samples expressed fascin, and higher fascin histoscores correlated with poor outcomes, vascular invasion, and time to recurrence. CONCLUSIONS: The actin-bundling protein fascin is regulated by slug and involved in late-stage PanIN and PDAC formation in mice. Fascin appears to promote formation of filopodia and invasive activities of PDAC cells. Its levels in human PDAC correlate with outcomes and time to recurrence, indicating it might be a marker or therapeutic target for pancreatic cancer.

Fascin regulates the migration of subventricular zone-derived neuroblasts in the postnatal brain.

After birth, stem cells in the subventricular zone (SVZ) generate neuroblasts that migrate along the rostral migratory stream (RMS) to become interneurons in the olfactory bulb (OB). This migration is a fundamental event controlling the proper integration of new neurons in a pre-existing synaptic network. Many regulators of neuroblast migration have been identified; however, still very little is known about the intracellular molecular mechanisms controlling this process. Here, we show that the actin-bundling protein fascin is highly upregulated in mouse SVZ-derived migratory neuroblasts. Fascin-1ko mice display an abnormal RMS and a smaller OB. Bromodeoxyuridine labeling experiments show that lack of fascin significantly impairs neuroblast migration, but does not appear to affect cell proliferation. Moreover, fascin depletion substantially alters the polarized morphology of rat neuroblasts. Protein kinase C (PKC)-dependent phosphorylation of fascin on Ser39 regulates its actin-bundling activity. In vivo postnatal electroporation of phosphomimetic (S39D) or nonphosphorylatable (S39A) fascin variants followed by time-lapse imaging of brain slices demonstrates that the phospho-dependent modulation of fascin activity ensures efficient neuroblast migration. Finally, fluorescence lifetime imaging microscopy studies in rat neuroblasts reveal that the interaction between fascin and PKC can be modulated by cannabinoid signaling, which controls neuroblast migration in vivo. We conclude that fascin, whose upregulation appears to mark the transition to the migratory neuroblast stage, is a crucial regulator of neuroblast motility. We propose that a tightly regulated phospho/dephospho-fascin cycle modulated by extracellular signals is required for the polarized morphology and migration in neuroblasts, thus contributing to efficient neurogenesis.

Microsatellite Instability Testing and Prognostic Implications in Colorectal Cancer.

Given the crucial predictive implications of microsatellite instability (MSI) in colorectal cancer (CRC), MSI screening is commonly performed in those with and at risk for CRC. Here, we compared results from immunohistochemistry (IHC) and the droplet digital PCR (ddPCR) MSI assay on formalin-fixed paraffin-embedded tumor samples from 48 patients who underwent surgery for colon and rectal cancer by calculating Cohen's kappa measurement (k), revealing high agreement between the methods (k = 0.915). We performed Kaplan-Meier survival analyses and univariate and multivariate Cox regression to assess the prognostic significance of ddPCR-based MSI and to identify clinicopathological features associated with CRC outcome. Patients with MSI-high had better overall survival (OS; p = 0.038) and disease-free survival (DFS; p = 0.049) than those with microsatellite stability (MSS). When stratified by primary tumor location, right-sided CRC patients with MSI-high showed improved DFS, relative to those with MSS (p < 0.001), but left-sided CRC patients did not. In multivariate analyses, MSI-high was associated with improved OS (hazard ratio (HR) = 0.221, 95% confidence interval (CI): 0.026-0.870, p = 0.042), whereas the loss of DNA mismatch repair protein MutL homolog 1 (MLH1) expression was associated with worse OS (HR = 0.133, 95% CI: 0.001-1.152, p = 0.049). Our results suggest ddPCR is a promising tool for MSI detection. Given the opposing effects of MSI-high and MLH1 loss on OS, both ddPCR and IHC may be complementary for the prognostic assessment of CRC.

Melatonin Increases Proliferation and Decreases Apoptosis of GC-1 spg Cells by Upregulating the Expression of circTec.

Melatonin has been shown to be beneficial for the motility of human sperm, although its mechanism remains to be uncovered. Circular RNAs (circRNAs) have been shown to regulate cellular function in many diseases. However, there has been no relevant research on the effect of melatonin on sperm circRNAs. In this study, we aimed to explore the changes in circRNAs after melatonin treatment of GC-1 spg cells and identify key functional circRNAs. The results showed that melatonin enhanced the proliferation and reduced the apoptosis of GC-1 spg cells. A total of 1423 circRNAs were found to be significantly differentially expressed between groups with and without melatonin treatment. Of these circRNAs, 702 were upregulated and 721 were downregulated. circTec was one of the upregulated circRNAs. Suppressing the expression of circTec significantly reduced cell proliferation and mammalian target of rapamycin (mTOR) signaling pathway activation but promoted melatonin-treated GC-1 spg cell apoptosis. In conclusion, melatonin increased the expression of circTec to exert its physiological effects on GC-1 spg cells, possibly by activating the mTOR signaling pathway. These results enhance our understanding of the biological function of circTec and its regulation by melatonin in spermatogenesis and infertility.

The use of cell free DNA (cfDNA) for mutational screening of multiple myeloma.

Multiple myeloma (MM) is an incurable haematological malignancy which relies heavily on bone marrow biopsies for disease monitoring and prediction of treatment response. In recent years, liquid biopsy derived cell-free DNA (cfDNA) has emerged as alternative for invasive biopsies. This pilot study aimed to evaluate the feasibility of using cfDNA for the detection of oncogenic mutations in the mitogen-activated protein kinase (MAPK) pathway genes NRAS, KRAS, and BRAF in MM patients. Matched peripheral blood and bone marrow aspirates were collected from thirteen MM patients at various disease stages. cfDNA was isolated using the Qiagen Circulating Nucleic Acid Kit while bone marrow DNA was extracted using the Maxwell Promega platform. The presence of NRAS, KRAS, and BRAF mutations was analysed by ddPCR and compared between the cfDNA and gDNA samples. Although our data come from a small patient cohort, mutations were detected, which supports cfDNA utility for mutational screening and prognostication in MM.

Circulating Tumour Cell Associated MicroRNA Profiles Change during Chemoradiation and Are Predictive of Response in Locally Advanced Rectal Cancer.

Locally advanced rectal cancer (LARC) has traditionally been treated with trimodality therapy consisting of neoadjuvant radiation +/- chemotherapy, surgery, and adjuvant chemotherapy. There is currently a clinical need for biomarkers to predict treatment response and outcomes, especially during neoadjuvant therapy. Liquid biopsies in the form of circulating tumour cells (CTCs) and circulating nucleic acids in particular microRNAs (miRNA) are novel, the latter also being highly stable and clinically relevant regulators of disease. We studied a prospective cohort of 52 patients with LARC, and obtained samples at baseline, during treatment, and post-treatment. We enumerated CTCs during chemoradiation at these three time-points, using the IsofluxTM (Fluxion Biosciences Inc., Alameda, CA, USA) CTC Isolation and detection platform. We then subjected the isolated CTCs to miRNA expression analyses, using a panel of 106 miRNA candidates. We identified CTCs in 73% of patients at baseline; numbers fell and miRNA expression profiles also changed during treatment. Between baseline and during treatment (week 3) time-points, three microRNAs (hsa-miR-95, hsa-miR-10a, and hsa-miR-16-1*) were highly differentially expressed. Importantly, hsa-miR-19b-3p and hsa-miR-483-5p were found to correlate with good response to treatment. The latter (hsa-miR-483-5p) was also found to be differentially expressed between good responders and poor responders. These miRNAs represent potential predictive biomarkers, and thus a potential miRNA-based treatment strategy. In this study, we demonstrate that CTCs are present and can be isolated in the non-metastatic early-stage cancer setting, and their associated miRNA profiles can potentially be utilized to predict treatment response.

From Pediatric to Adult Brain Cancer: Exploring Histone H3 Mutations in Australian Brain Cancer Patients.

Genetic histone variants have been implicated in cancer development and progression. Mutations affecting the histone 3 (H3) family, H3.1 (encoded by HIST1H3B and HIST1H3C) and H3.3 (encoded by H3F3A), are mainly associated with pediatric brain cancers. While considered poor prognostic brain cancer biomarkers in children, more recent studies have reported H3 alterations in adult brain cancer as well. Here, we established reliable droplet digital PCR based assays to detect three histone mutations (H3.3-K27M, H3.3-G34R, and H3.1-K27M) primarily linked to childhood brain cancer. We demonstrate the utility of our assays for sensitively detecting these mutations in cell-free DNA released from cultured diffuse intrinsic pontine glioma (DIPG) cells and in the cerebral spinal fluid of a pediatric patient with DIPG. We further screened tumor tissue DNA from 89 adult patients with glioma and 1 with diffuse hemispheric glioma from Southwestern Sydney, Australia, an ethnically diverse region, for these three mutations. No histone mutations were detected in adult glioma tissue, while H3.3-G34R presence was confirmed in the diffuse hemispheric glioma patient.

Melatonin enhances spermatogonia activity through promoting KIAA1429-mediated m6A deposition to activate the PI3K/AKT signaling.

Melatonin is a key neuroendocrine hormone that promotes spermatogenesis and sperm motility, but the underlying mechanisms remains poorly understood. In this study, we aimed to investigate the possible roles of m6A (N6--methyl-adenosine) in mediating melatonin-regulated spermatogonia activity alterations. In this study, mouse-derived GC-1 spermatogonia (spg) cell line was used as the in vitro cellular model. The viability, proliferation rates and apoptosis of spermatogonia were detected via CCK-8, Edu staining and flow cytometry respectively. Total m6A level was quantitated by dot blot, while mRNA and proteins contents in spermatogonia were measured by qRT-PCR and western blot respectively. Differentially expressed mRNAs were characterized by deep RNA sequencing method. Results showed that melatonin significantly promoted viability and proliferation rate while inhibited apoptosis in the GC-1 spg cells. The total m6A levels in GC-1 spg cells were also greatly increased by melatonin treatment, accompanied by remarkable expressional elevation of the m6A writer KIAA1429. Moreover, the regulation of GC-1 spg cell viability, proliferation and apoptosis by melatonin were greatly abrogated by KIAA1429 silencing but effectively strengthened by KIAA1429 overexpression. In addition, KIAA1429 overexpression regulates multiple biological process and signaling pathways in spermatogonia such as the PI3K/AKT signaling. The PI3K inhibitor LY294002 effectively mitigated the regulation of spermatogonia activity by KIAA1429 overexpression under melatonin treatment. Taken together, melatonin promotes spermatogonia activity via enhancing KIAA1429 expression and m6A RNA methylation to activate the downstream PI3K/AKT signaling pathway.

Biomarkers of Castrate Resistance in Prostate Cancer: Androgen Receptor Amplification and T877A Mutation Detection by Multiplex Droplet Digital PCR.

Androgen Receptor (AR) alterations (amplification, point mutations, and splice variants) are master players in metastatic castration resistant prostate cancer (CRPC) progression and central therapeutic targets for patient management. Here, we have developed two multiplexed droplet digital PCR (ddPCR) assays to detect AR copy number (CN) and the key point mutation T877A. Overcoming challenges of determining gene amplification from liquid biopsies, these assays cross-validate each other to produce reliable AR amplification and mutation data from plasma cell free DNA (cfDNA) of advanced prostate cancer (PC) patients. Analyzing a mixed PC patient cohort consisting of CRPC and hormone sensitive prostate cancer (HSPC) patients showed that 19% (9/47) patients had AR CN amplification. As expected, only CRPC patients were positive for AR amplification, while interestingly the T877A mutation was identified in two patients still considered HSPC at the time. The ddPCR based analysis of AR alterations in cfDNA is highly economic, feasible, and informative to provide biomarker detection that may help to decide on the best follow-up therapy for CRPC patients.

Prognostic and Predictive Value of Liquid Biopsy-Derived Androgen Receptor Variant 7 (AR-V7) in Prostate Cancer: A Systematic Review and Meta-Analysis.

In advanced prostate cancer, access to recent diagnostic tissue samples is restricted and this affects the analysis of the association of evolving biomarkers such as AR-V7 with metastatic castrate resistance. Liquid biopsies are emerging as alternative analytes. To clarify clinical value of AR-V7 detection from liquid biopsies, here we performed a meta-analysis on the prognostic and predictive value of androgen receptor variant 7 (AR-V7) detected from liquid biopsy for patients with prostate cancer (PC), three databases, the Embase, Medline, and Scopus were searched up to September 2021. A total of 37 studies were included. The effects of liquid biopsy AR-V7 status on overall survival (OS), radiographic progression-free survival (PFS), and prostate-specific antigen (PSA)-PFS were calculated with RevMan 5.3 software. AR-V7 positivity detected in liquid biopsy significantly associates with worse OS, PFS, and PSA-PFS (P <0.00001). A subgroup analysis of patients treated with androgen receptor signaling inhibitors (ARSi such as abiraterone and enzalutamide) showed a significant association of AR-V7 positivity with poorer OS, PFS, and PSA-PFS. A statistically significant association with OS was also found in taxane-treated patients (P = 0.04), but not for PFS (P = 0.21) or PSA-PFS (P = 0.93). For AR-V7 positive patients, taxane treatment has better OS outcomes than ARSi (P = 0.01). Study quality, publication bias and sensitivity analysis were integrated in the assessment. Our data show that liquid biopsy AR-V7 is a clinically useful biomarker that is associated with poor outcomes of ARSi-treated castrate resistant PC (CRPC) patients and thus has the potential to guide patient management and also to stratify patients for clinical trials. More studies on chemotherapy-treated patients are warranted. Systematic Review Registration: PROSPERO, CRD42021239353.