Molecular mechanisms, targets and clinical potential of berberine in regulating metabolism: a review focussing on databases and molecular docking studies.

Background: Metabolic imbalance is the common basis of many diseases. As natural isoquinoline alkaloid, berberine (BBR) has shown great promise in regulating glucose and lipids metabolism and treating metabolic disorders. However, the related mechanism still lacks systematic research. Aim: To discuss the role of BBR in the whole body's systemic metabolic regulation and further explore its therapeutic potential and targets. Method: Based on animal and cell experiments, the mechanism of BBR regulating systemic metabolic processes is reviewed. Potential metabolism-related targets were summarized using Therapeutic Target Database (TTD), DrugBank, GeneCards, and cutting-edge literature. Molecular modeling was applied to explore BBR binding to the potential targets. Results: BBR regulates the whole-body metabolic response including digestive, circulatory, immune, endocrine, and motor systems through adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK)/mammalian target of rapamycin (mTOR), sirtuin (SIRT)1/forkhead box O (FOXO)1/sterol regulatory element-binding protein (SREBP)2, nuclear factor erythroid 2-related factor (Nrf) 2/heme oxygenase (HO)-1, and other signaling pathways. Through these reactions, BBR exerts hypoglycemic, lipid-regulating, anti-inflammatory, anti-oxidation, and immune regulation. Molecular docking results showed that BBR could regulate metabolism targeting FOXO3, Nrf2, NAD(P)H quinone oxidoreductase 1 (NQO1), glutathione peroxidase (Gpx) 4 and phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA). Evaluating the target clinical effects, we found that BBR has the therapeutic potential of anti-aging, anti-cancer, relieving kidney disease, regulating the nervous system, and alleviating other chronic diseases. Conclusion: This review elucidates the interaction between potential targets and small molecular metabolites by exploring the mechanism of BBR regulating metabolism. That will help pharmacologists to identify new promising metabolites interacting with these targets.

Herbal medicine for Hashimoto's thyroiditis: A systematic review and network meta-analysis.

ETHNOPHARMACOLOGICAL RELEVANCE: Conventional treatments for Hashimoto's thyroiditis (HT) are limited. Herbal medicines (HM) are considered a potential intervention for the treatment of HT. AIM OF THE STUDY: This study aimed to investigate the efficacy and safety of HM for HT. MATERIALS AND METHODS: A Bayesian network meta-analysis was conducted for patients with HT in randomized controlled trials identified in PubMed, Cochrane Library, Web of Science, EMBASE, Chinese Clinical Trial Registry (Chi CTR), China National Knowledge Infrastructure (CNKI), China Science and Technology Journal Database (the VIP), China Chinese Biomedical Database (CBM), and Wanfang Database were searched from their inception to Oct 1, 2022. Outcomes included the primary outcome (TPOAb), secondary outcomes (TSH, TGAb, FT3, FT4, and traditional Chinese medicine symptom scores), and adverse events. This study was registered in PROSPERO (CRD42022363640). RESULTS: Sixteen trials were reviewed and 16 HM formulae were compared. Compared with non-drug therapy (NDT), all therapies, except for Tiaoqi-Qingjie Therapy, reduced the primary outcome of TPOAb with different levels of effectiveness, ranging from 0.01 (95%CI 0.00, 0.02) to 0.92 (95%CI 0.56, 1.53). Ranking probability analysis indicated that Yiqi Huayu Recipe, Liqi Xiaoying decoction, and Shugan Sanjie therapy reduced thyroid antibody levels the most, including TPOAb (100.0%, 90.9%, and 90.3%, respectively) and TGAb (98.3%, 94.4%, and 87.3%, respectively). All HMs displayed a significant effect on the TCM Symptom score and possibly benefitted the treatment of HT, ranging from 6.62 (95% CI 2.06, 21.24) to 94.50 (95% CI 15.97, 559.14). No serious adverse events were reported. CONCLUSIONS: Herbal medicines may be effective in the treatment of HT, especially in reducing thyroid antibody levels and improving clinical symptoms without affecting thyroid function. However, these results should be considered preliminary and further verified using high-quality evidence.

Clinical efficacy and molecular mechanism of Chinese medicine in the treatment of autoimmune thyroiditis.

ETHNOPHARMACOLOGICAL RELEVANCE: Autoimmune Thyroiditis (AIT) is a common refractory autoimmune disease of the endocrine system that may eventually lead to complete loss of thyroid function, with subsequent severe effects on the metabolism. Because of the deficiency in current clinical management of AIT, the need for alternative therapies is highlighted. With its multi-component and multi-target characteristics, Chinese medicine has good potential as an alternative therapy for AIT. AIM OF THE STUDY: The aim of this study was to systematically summarize the clinical efficacy and safety evaluation of TCM and its active ingredients in the treatment and regulation of AIT. Additionally, we provide an in-depth discussion of the relevant mechanisms and molecular targets to understand the protective effects of traditional Chinese medicine on AIT and explore new ideas for clinical treatment. MATERIALS AND METHODS: The literature related to "Hashimoto", "autoimmune thyroiditis", "traditional Chinese medicine," and "Chinese herbal medicine" was systematically summarized and reviewed from Web of Science Core Collection, PubMed, CNKI, and other databases. Domestic and international literature were analyzed, compared, and reviewed. RESULTS: An increasing number of studies have demonstrated that herbal medicines can intervene in immunomodulation, with pharmacological effects such as antibody lowering, anti-inflammatory, anti-apoptotic thyroid follicular cells, regulation of intestinal flora, and regulation of estrogen and progesterone levels. The signaling pathways and molecular targets of the immunomodulatory effects of Chinese herbal medicine for AIT may include Fas/FasL, Caspase, BCL-2, and TLRs/MyD88/NF-κB et al. CONCLUSIONS: The use of Chinese herbs in the treatment and management of AIT is clinically experienced, satisfactory, and safe. Future studies may evaluate the influence of herbal medicines on the occurrence and development of AIT by modulating the interaction between immune factors and conventional signaling pathways.

Evaluating niraparib versus active symptom control in patients with previously treated mesothelioma (NERO): a study protocol for a multicentre, randomised, two-arm, open-label phase II trial in UK secondary care centres.

BACKGROUND: Malignant mesothelioma is a rapidly lethal cancer that has been increasing at an epidemic rate over the last three decades. Targeted therapies for mesothelioma have been lacking. A previous study called MiST1 (NCT03654833), evaluated the efficacy of Poly (ADP-ribose) polymerase (PARP) inhibition in mesothelioma. This study met its primary endpoint with 15% of patients having durable responses exceeding 1 year. Therefore, there is a need to evaluate PARP inhibitors in relapsed mesothelioma patients, where options are limited. Niraparib is the PARP inhibitor used in NERO. METHODS: NERO is a multicentre, two-arm, open-label UK randomised phase II trial designed to evaluate the efficacy of PARP inhibition in relapsed mesothelioma. 84 patients are being recruited. NERO is not restricted by line of therapy; however, eligible participants must have been treated with an approved platinum based systemic therapy. Participants will be randomised 2:1, stratified according to histology and response to prior platinum-based chemotherapy, to receive either active symptom control (ASC) and niraparib or ASC alone, for up to 24 weeks. Participants will be treated until disease progression, withdrawal, death or development of significant treatment limiting toxicity. Participants randomised to niraparib will receive 200 or 300 mg daily in a 3-weekly cycle. The primary endpoint is progression-free survival, where progression is determined by modified Response Evaluation Criteria in Solid Tumors (mRECIST) or RECIST 1.1; investigator reported progression; or death from any cause, whichever comes first. Secondary endpoints include overall survival, best overall response, 12-week and 24 week disease control, duration of response, treatment compliance and safety/tolerability. If NERO shows niraparib to be safe and biologically effective, it may lead to future late phase randomised controlled trials in relapsed mesothelioma. ETHICS AND DISSEMINATION: The study received ethical approval from London-Hampstead Research Ethics Committee on 06-May-2022 (22/LO/0281). Data from all centres will be analysed together and published as soon as possible. TRIAL REGISTRATION NUMBER: ISCRTN16171129; NCT05455424.

Jiedutongluotiaogan formula restores pancreatic function by suppressing excessive autophagy and endoplasmic reticulum stress.

CONTEXT: Jiedutongluotiaogan formula (JTTF), a traditional Chinese medicine (TCM), could promote islet function. However, the potential effect of JTTF on endoplasmic reticulum stress (ERS) and autophagy have not been reported. OBJECTIVE: This study explores the potential effect of JTTF on ERS and autophagy in the pancreas. MATERIALS AND METHODS: The Zucker diabetic fatty (ZDF) rats were randomised into five groups, control, model, JTTF (1, 3, 5 g/kg/day for 12 weeks). LPS induced pancreatic ß-cells were treated with JTTF (50, 100, 200 µg/mL). LPS was used to induce pancreatic ß-cell injury, with cell viability and insulin secretion evaluated using MTT, glucose-stimulated insulin secretion (GSIS) assays, and PCR. Intracellular Ca2+ concentration was measured using flow cytometry, while ERS and autophagy levels were monitored via Western blotting and/or immunostaining. RESULTS: Compared with the model group, body weight, FGB, HbA1c, IPGTT, FINs, and HOMA-IR in JTTF treatment groups were significantly reduced. In islets cells treated with JTTF, the pancreatic islet cells in the JTTF group were increased, lipid droplets were reduced, and there was a decrease in Ca2+ (16.67%). After JTTF intervention, PERK, p-PERK, IRE1α, p- IRE1α, ATF6, eIF2α, GRP78, p-ULK1, LC3 and p62 expression decreased, whereas Beclin1and p-mTOR expression increased. In addition, the expression of proteins related to apoptosis in the JTTF groups were lower than those in the control group. DISCUSSION AND CONCLUSIONS: JTTF may alleviate pancreatic ß-cell injury by inhibiting ER stress and excessive autophagy in diabetic rats. This provides a new direction for treating diabetes and restoring pancreatic dysfunction by TCM.

Pharmacological effects of polydatin in the treatment of metabolic diseases: A review.

BACKGROUND: Metabolic diseases (MDs), a series of chronic disorders, severely decreases the quality of life for patients but also cause a heavy economic burden. Emerging evidence suggests that Polydatin (PD), an important glucoside of resveratrol, is widely distributed in many plants and has shown good therapeutic potential in metabolic diseases. PURPOSE: To review the PD discovered before 2021 and their potential to treat metabolic diseases. The activities against diabetes, Obesity, atherosclerosis, NAFLD, NASH, hyperlipidemia, and gout with special emphasis on pharmacology, pharmacokinetics, mechanisms of action, possible roles in current medicine, and future perspectives are discussed. METHODS: A comprehensive search of published literature was conducted to locate original publications pertaining to polydatin and MDs through the end of 2021 using MEDLINE, Elsevier, Springer, PubMed, Scholar, and CNKI databases. The main inquiry used was for the presence of the following keywords in various combinations in the abstracts: 'Polydatin', 'Metabolic diseases', 'Pharmacology', 'Toxicology', 'Pharmacokinetics', 'Diabetes', 'Obesity', 'Atherosclerosis', 'Non-alcoholic fatty liver disease', 'Non-alcoholic steatohepatitis', 'Hyperlipidemia', and 'Gout'. RESULTS: The search yielded 987 articles, of which 33 articles were included in this review. Studies have revealed that PD can promote insulin secretion, alleviate insulin resistance, regulate glucose and lipid metabolism, reduce liver lipid deposition, inhibit inflammation, oxidative stress, and decrease uric acid deposition in preclinical experiments. The underlying mechanisms of PD in treatment MDs may be attributed to the regulation of multiple signaling pathways, including. NF-κB, AGEs/RAGE, MAPK/ERK, AMPK/LDLR, IRS1/PI3K/AKT, LKB1/AMPK, PPARß-NO, SIRT1-PGC-1α-SOD2, PKC, etc., The pharmacokinetic profiles of PD provide valuable information on therapeutic efficacy in treating metabolic diseases. CONCLUSION: This review summarizes the available reports and evidence which support the use of PD as a potential candidate in the treatment of MDs and provides an overview of the modulatory effects of PD in metabolic diseases and cell signaling pathways, which may have important implications in its future clinical use.

Treatment of COVID-19 in Hemodialysis Patients Using Traditional Chinese Medicine: A Single-Center, Retrospective Study.

Background: To explore the effect of combining traditional Chinese medicine (TCM) and Western medicine in hemodialysis patients with coronavirus disease 2019 (COVID-19). Methods: This study was conducted from 27 January 2020 to 17 March 2020 in Wuhan Third Hospital Guanggu Branch, Wuhan, China. Fifty-three patients were included and divided into a control group (CG), which received Western medicine and a combined treatment group, which received TCM and Western medicine (TG). Clinical and laboratory data, TCM symptom scores, and chest computed tomography results were extracted and compared between the two groups. Results: The TG included 21 (67.7%) men and 10 (32.3%) women with a mean age of 61.02 (standard deviation [SD] 15.07, range 26-89) years. The mean dialysis duration in the TG was 49 (SD 31) months. Of all patients in the TG, 27 (87.1%) had fatigue, 18 (58.1%) had dry cough, 16 (51.6%) had anorexia, 11 (35.5%) had dyspnea, and 11 (35.5%) had fever. The CG included 14 (63.6%) men and 8 (36.4%) women with a mean age of 61.45 (SD 13.78, range 36-84) years. The mean dialysis duration in the CG was 63 (SD 46) months. Of all patients in the CG, 21 (95.5%) had fatigue, 12 (54.5%) had dry cough, 17 (77.3%) had anorexia, 12 (54.5%) had dyspnea, and 7 (31.8%) had fever. After treatment, the TCM symptom scores of the two groups decreased; the anorexia scores were lower in the TG than in the CG (p < 0.05). After treatment, albumin increased and D-dimer, C-reactive protein, and lactate dehydrogenase levels decreased in the TG. The d-dimer levels were lower and the albumin level was higher in the TG than in the CG after treatment (p < 0.05). The cure rate was higher, and the mortality rate was lower in the TG than in the CG (p < 0.05). Conclusion: A combination of TCM and Western medicine in hemodialysis patients with COVID-19 could relieve symptoms and help recovery. Further evidence from larger randomized controlled trials is needed to confirm our results.

Identifying Biomarkers from Transcriptomic Signatures in Renal Allograft Biopsies Using Deceased and Living Donors.

The survival of transplant kidneys using deceased donors (DD) is inferior to living donors (LD). In this study, we conducted a whole-transcriptome expression analysis of 24 human kidney biopsies paired at 30 minutes and 3 months post-transplantation using DD and LD. The transcriptome profile was found significantly different between two time points regardless of donor types. There were 446 differentially expressed genes (DEGs) between DD and LD at 30 minutes and 146 DEGs at 3 months, with 25 genes common to both time points. These DEGs reflected donor injury and acute immune responses associated with inflammation and cell death as early as at 30 minutes, which could be a precious window of potential intervention. DEGs at 3 months mainly represented the changes of adaptive immunity, immunosuppressive treatment, remodeling or fibrosis via different networks and signaling pathways. The expression levels of 20 highly DEGs involved in kidney diseases and 10 genes dysregulated at 30 minutes were found correlated with renal function and histology at 12 months, suggesting they could be potential biomarkers. These genes were further validated by quantitative polymerase chain reaction (qPCR) in 24 samples analysed by microarray, as well as in a validation cohort of 33 time point unpaired allograft biopsies. This analysis revealed that SERPINA3, SLPI and CBF were up-regulated at 30 minutes in DD compared to LD, while FTCD and TASPN7 were up-regulated at both time points. At 3 months, SERPINA3 was up-regulated in LD, but down-regulated in DD, with increased VCAN and TIMP1, and decreased FOS, in both donors. Taken together, divergent transcriptomic signatures between DD and LD, and changed by the time post-transplantation, might contribute to different allograft survival of two type kidney donors. Some DEGs including FTCD and TASPN7 could be novel biomarkers not only for timely diagnosis, but also for early precise genetic intervention at donor preservation, implantation and post-transplantation, in particular to effectively improve the quality and survival of DD.

Transcriptional perturbation of protein arginine methyltransferase-5 exhibits MTAP-selective oncosuppression.

We hypothesized that small molecule transcriptional perturbation could be harnessed to target a cellular dependency involving protein arginine methyltransferase 5 (PRMT5) in the context of methylthioadenosine phosphorylase (MTAP) deletion, seen frequently in malignant pleural mesothelioma (MPM). Here we show, that MTAP deletion is negatively prognostic in MPM. In vitro, the off-patent antibiotic Quinacrine efficiently suppressed PRMT5 transcription, causing chromatin remodelling with reduced global histone H4 symmetrical demethylation. Quinacrine phenocopied PRMT5 RNA interference and small molecule PRMT5 inhibition, reducing clonogenicity in an MTAP-dependent manner. This activity required a functional PRMT5 methyltransferase as MTAP negative cells were rescued by exogenous wild type PRMT5, but not a PRMT5E444Q methyltransferase-dead mutant. We identified c-jun as an essential PRMT5 transcription factor and a probable target for Quinacrine. Our results therefore suggest that small molecule-based transcriptional perturbation of PRMT5 can leverage a mutation-selective vulnerability, that is therapeutically tractable, and has relevance to 9p21 deleted cancers including MPM.

Clonal architecture in mesothelioma is prognostic and shapes the tumour microenvironment.

Malignant Pleural Mesothelioma (MPM) is typically diagnosed 20-50 years after exposure to asbestos and evolves along an unknown evolutionary trajectory. To elucidate this path, we conducted multi-regional exome sequencing of 90 tumour samples from 22 MPMs acquired at surgery. Here we show that exomic intratumour heterogeneity varies widely across the cohort. Phylogenetic tree topology ranges from linear to highly branched, reflecting a steep gradient of genomic instability. Using transfer learning, we detect repeated evolution, resolving 5 clusters that are prognostic, with temporally ordered clonal drivers. BAP1/-3p21 and FBXW7/-chr4 events are always early clonal. In contrast, NF2/-22q events, leading to Hippo pathway inactivation are predominantly late clonal, positively selected, and when subclonal, exhibit parallel evolution indicating an evolutionary constraint. Very late somatic alteration of NF2/22q occurred in one patient 12 years after surgery. Clonal architecture and evolutionary clusters dictate MPM inflammation and immune evasion. These results reveal potentially drugable evolutionary bottlenecking in MPM, and an impact of clonal architecture on shaping the immune landscape, with potential to dictate the clinical response to immune checkpoint inhibition.

The Circulating Nucleic Acid Characteristics of Non-Metastatic Soft Tissue Sarcoma Patients.

Soft tissue sarcomas (STS) are rare, malignant tumours with a generally poor prognosis. Our aim was to explore the potential of cell free DNA (cfDNA) and circulating tumour DNA (ctDNA) analysis to track non-metastatic STS patients undergoing attempted curative treatment. The analysed cohort (n = 29) contained multiple STS subtypes including myxofibrosarcomas, undifferentiated pleomorphic sarcomas, leiomyosarcomas, and dedifferentiated liposarcomas amongst others. Perioperative cfDNA levels trended towards being elevated in patients (p = 0.07), although did not correlate with tumour size, grade, recurrence or subtype, suggesting a limited diagnostic or prognostic role. To characterise ctDNA, an amplicon panel covering three genes commonly mutated in STSs was first trialled on serial plasma collected from nine patients throughout follow-up. This approach only identified ctDNA in 2.5% (one in 40) of the analysed samples. Next custom-designed droplet digital PCR assays and Ion AmpliSeq™ panels were developed to track single nucleotide variants identified in patients' STSs by whole exome sequencing (1-6 per patient). These approaches identified ctDNA in 17% of patients. Although ctDNA was identified before radiologically detectable recurrence in two cases, the absence of demonstrable ctDNA in 83% of cases highlights the need for much work before circulating nucleic acids can become a useful means to track STS patients.

Fibroblast-Derived STC-1 Modulates Tumor-Associated Macrophages and Lung Adenocarcinoma Development.

The tumor microenvironment (TME) consists of different cell types, including tumor-associated macrophages (TAMs) and tumor-associated fibroblasts (TAFs). How these cells interact and contribute to lung carcinogenesis remains elusive. Using G12DKRAS- and V600EBRAF-driven mouse lung models, we identify the pleiotropic glycoprotein stanniocalcin-1 (STC1) as a regulator of TAM-TAF interactions. STC1 is secreted by TAFs and suppresses TAM differentiation, at least in part, by sequestering the binding of GRP94, an autocrine macrophage-differentiation-inducing factor, to its cognate scavenger receptors. The accumulation of mature TAMs in the Stc1-deficient lung leads to enhanced secretion of TGF-ß1 and, thus, TAF accumulation in the TME. Consistent with the mouse data, in human lung adenocarcinoma, STC1 expression is restricted to myofibroblasts, and a significant increase of naive macrophages is detected in STC1-high compared with STC1-low cases. This work increases our understanding of lung adenocarcinoma development and suggests new approaches for therapeutic targeting of the TME.

Utilizing network pharmacology to explore the underlying mechanism of Radix Salviae in diabetic retinopathy.

INTRODUCTION: Radix Salviae (Dan-shen in pinyin), a classic Chinese herb, has been extensively used to treat diabetic retinopathy in clinical practice in China for many years. However, the pharmacological mechanisms of Radix Salviae remain vague. The aim of this study was to decrypt the underlying mechanisms of Radix Salviae in the treatment of diabetic retinopathy using a systems pharmacology approach. METHODS: A network pharmacology-based strategy was proposed to elucidate the underlying multi-component, multi-target, and multi-pathway mode of action of Radix Salviae against diabetic retinopathy. First, we collected putative targets of Radix Salviae based on the Traditional Chinese Medicine System Pharmacology database and a network of the interactions among the putative targets of Radix Salviae and known therapeutic targets of diabetic retinopathy was built. Then, two topological parameters, "degree" and "closeness certainty" were calculated to identify the major targets in the network. Furthermore, the major hubs were imported to the Database for Annotation, Visualization and Integrated Discovery to perform a pathway enrichment analysis. RESULTS: A total of 130 nodes, including 18 putative targets of Radix Salviae, were observed to be major hubs in terms of topological importance. The results of pathway enrichment analysis indicated that putative targets of Radix Salviae mostly participated in various pathways associated with angiogenesis, protein metabolism, inflammatory response, apoptosis, and cell proliferation. The putative targets of Radix Salviae (vascular endothelial growth factor, matrix metalloproteinases, plasminogen, insulin-like growth factor-1, and cyclooxygenase-2) were recognized as active factors involved in the main biological functions of treatment, which implied that these were involved in the underlying mechanisms of Radix Salviae on diabetic retinopathy. CONCLUSIONS: Radix Salviae could alleviate diabetic retinopathy via the molecular mechanisms predicted by network pharmacology. This research demonstrates that the network pharmacology approach can be an effective tool to reveal the mechanisms of traditional Chinese medicine from a holistic perspective.

Sensitivity of Colorectal Cancer to Arginine Deprivation Therapy is Shaped by Differential Expression of Urea Cycle Enzymes.

Tumors deficient in the urea cycle enzymes argininosuccinate synthase-1 (ASS1) and ornithine transcarbamylase (OTC) are unable to synthesize arginine and can be targeted using arginine-deprivation therapy. Here, we show that colorectal cancers (CRCs) display negligible expression of OTC and, in subset of cases, ASS1 proteins. CRC cells fail to grow in arginine-free medium and dietary arginine deprivation slows growth of cancer cells implanted into immunocompromised mice. Moreover, we report that clinically-formulated arginine-degrading enzymes are effective anticancer drugs in CRC. Pegylated arginine deiminase (ADI-PEG20), which degrades arginine to citrulline and ammonia, affects growth of ASS1-negative cells, whereas recombinant human arginase-1 (rhArg1peg5000), which degrades arginine into urea and ornithine, is effective against a broad spectrum of OTC-negative CRC cell lines. This reflects the inability of CRC cells to recycle citrulline and ornithine into the urea cycle. Finally, we show that arginase antagonizes chemotherapeutic drugs oxaliplatin and 5-fluorouracil (5-FU), whereas ADI-PEG20 synergizes with oxaliplatin in ASS1-negative cell lines and appears to interact with 5-fluorouracil independently of ASS1 status. Overall, we conclude that CRC is amenable to arginine-deprivation therapy, but we warrant caution when combining arginine deprivation with standard chemotherapy.

Circulating tumour-derived DNA in metastatic soft tissue sarcoma.

Following treatment 40% of soft tissue sarcoma (STS) patients suffer disease recurrence. In certain cancers circulating cell free DNA (cfDNA) and circulating tumour-derived DNA (ctDNA) characteristics correlate closely with disease burden, making them exciting potential sources of biomarkers. Despite this, the circulating nucleic acid characteristics of only 2 STS patients have been reported to date. To address this we used an Ion AmpliSeq™ panel custom specifically designed for STS patients to conduct a genetic characterisation of plasma cfDNA, buffy coat (germline) DNA and where available Formalin-Fixed Paraffin-Embedded (FFPE) primary STS tissue DNA in a cohort of 11 metastatic STS patients. We found that total cfDNA levels were significantly elevated in the STS patients analysed, and weakly correlated with disease burden. Using our Ion AmpliSeq™ panel we also successfully detected ctDNA in 4/11 (36%) patients analysed with a wide variety of STS subtypes and disease burdens. This evidence included the presence of cancer associated TP53 / PIK3CA mutations in 2 patients' plasma and matched primary STS tumour tissue, and in the plasma alone for 2 patients. We also identified 2 potential examples of allelic loss of heterozygosity in an additional patient's STS DNA and cfDNA. This is the largest study performed characterising STS patient cfDNA/ctDNA and confirms that the field remains an attractive potential source of novel STS biomarkers. Further work is required to investigate the circulating nucleic acid characteristics of individual STS subtypes, and the potential prognostic or therapeutic roles that cfDNA/ctDNA may hold for patients with these complex tumours.

Over-expressed, N-terminally truncated BRAF is detected in the nucleus of cells with nuclear phosphorylated MEK and ERK.

BRAF is a cytoplasmic protein kinase, which activates the MEK-ERK signalling pathway. Deregulation of the pathway is associated with the presence of BRAF mutations in human cancer, the most common being V600E BRAF, although structural rearrangements, which remove N-terminal regulatory sequences, have also been reported. RAF-MEK-ERK signalling is normally thought to occur in the cytoplasm of the cell. However, in an investigation of BRAF localisation using fluorescence microscopy combined with subcellular fractionation of Green Fluorescent Protein (GFP)-tagged proteins expressed in NIH3T3 cells, surprisingly, we detected N-terminally truncated BRAF (ΔBRAF) in both nuclear and cytoplasmic compartments. In contrast, ΔCRAF and full-length, wild-type BRAF (WTBRAF) were detected at lower levels in the nucleus while full-length V600EBRAF was virtually excluded from this compartment. Similar results were obtained using ΔBRAF tagged with the hormone-binding domain of the oestrogen receptor (hbER) and with the KIAA1549-ΔBRAF translocation mutant found in human pilocytic astrocytomas. Here we show that GFP-ΔBRAF nuclear translocation does not involve a canonical Nuclear Localisation Signal (NLS), but is suppressed by N-terminal sequences. Nuclear GFP-ΔBRAF retains MEK/ERK activating potential and is associated with the accumulation of phosphorylated MEK and ERK in the nucleus. In contrast, full-length GFP-WTBRAF and GFP-V600EBRAF are associated with the accumulation of phosphorylated ERK but not phosphorylated MEK in the nucleus. These data have implications for cancers bearing single nucleotide variants or N-terminal deleted structural variants of BRAF.

Profiling tumour heterogeneity through circulating tumour DNA in patients with pancreatic cancer.

The majority of pancreatic ductal adenocarcinomas (PDAC) are diagnosed late so that surgery is rarely curative. Earlier detection could significantly increase the likelihood of successful treatment and improve survival. The aim of the study was to provide proof of principle that point mutations in key cancer genes can be identified by sequencing circulating free DNA (cfDNA) and that this could be used to detect early PDACs and potentially, premalignant lesions, to help target early effective treatment. Targeted next generation sequencing (tNGS) analysis of mutation hotspots in 50 cancer genes was conducted in 26 patients with PDAC, 14 patients with chronic pancreatitis (CP) and 12 healthy controls with KRAS status validated by digital droplet PCR. A higher median level of total cfDNA was observed in patients with PDAC (585 ng/ml) compared to either patients with CP (300 ng/ml) or healthy controls (175 ng/ml). PDAC tissue showed wide mutational heterogeneity, whereas KRAS was the most commonly mutated gene in cfDNA of patients with PDAC and was significantly associated with a poor disease specific survival (p=0.018). This study demonstrates that tNGS of cfDNA is feasible to characterise the circulating genomic profile in PDAC and that driver mutations in KRAS have prognostic value but cannot currently be used to detect early emergence of disease. Importantly, monitoring total cfDNA levels may have utility in individuals "at risk" and warrants further investigation.

Mutation Analysis of Cell-Free DNA and Single Circulating Tumor Cells in Metastatic Breast Cancer Patients with High Circulating Tumor Cell Counts.

PURPOSE: The purpose of this study was to directly compare mutation profiles in multiple single circulating tumor cells (CTC) and cell-free DNA (cfDNA) isolated from the same blood samples taken from patients with metastatic breast cancer (MBC). We aimed to determine whether cfDNA would reflect the heterogeneity observed in 40 single CTCs. EXPERIMENTAL DESIGN: CTCs were enumerated by CELLSEARCH. CTC count was compared with the quantity of matched cfDNA and serum CA15-3 and alkaline phosphatase (ALP) in 112 patients with MBC. In 5 patients with ≥100 CTCs, multiple individual EpCAM-positive CTCs were isolated by DEPArray and compared with matched cfDNA and primary tumor tissue by targeted next-generation sequencing (NGS) of about 2,200 mutations in 50 cancer genes. RESULTS: In the whole cohort, total cfDNA levels and cell counts (≥5 CTCs) were both significantly associated with overall survival, unlike CA15-3 and ALP. NGS analysis of 40 individual EpCAM-positive CTCs from 5 patients with MBC revealed mutational heterogeneity in PIK3CA, TP53, ESR1, and KRAS genes between individual CTCs. In all 5 patients, cfDNA profiles provided an accurate reflection of mutations seen in individual CTCs. ESR1 and KRAS gene mutations were absent from primary tumor tissue and therefore likely either reflect a minor subclonal mutation or were acquired with disease progression. CONCLUSIONS: Our results demonstrate that cfDNA reflects persisting EpCAM-positive CTCs in patients with high CTC counts and therefore may enable monitoring of the metastatic burden for clinical decision-making. Clin Cancer Res; 23(1); 88-96. ©2016 AACR.

Next Generation Sequencing of Circulating Cell-Free DNA for Evaluating Mutations and Gene Amplification in Metastatic Breast Cancer.

BACKGROUND: Breast cancer tissues are heterogeneous and show diverse somatic mutations and somatic copy number alterations (CNAs). We used a novel targeted next generation sequencing (NGS) panel to examine cell-free DNA (cfDNA) to detect somatic mutations and gene amplification in women with metastatic breast cancer (MBC). METHODS: cfDNA from pretreated patients (n = 42) and 9 healthy controls were compared with matched lymphocyte DNA by NGS, using a custom 158 amplicon panel covering hot-spot mutations and CNAs in 16 genes, with further validation of results by droplet digital PCR. RESULTS: No mutations were identified in cfDNA of healthy controls, whereas exactly half the patients with metastatic breast cancer had at least one mutation or amplification in cfDNA (mean 2, range 1-6) across a total of 13 genes. Longitudinal follow up showed dynamic changes to mutations and gene amplification in cfDNA indicating clonal and subclonal response to treatment that was more dynamic than cancer antigen 15-3 (CA15-3). Interestingly, at the time of blood sampling disease progression was occurring in 7 patients with erb-b2 receptor tyrosine kinase 2 (ERBB2) gene amplification in their cfDNA and 3 of these patients were human epidermal growth factor receptor 2 (HER2) negative at diagnosis, suggesting clonal evolution to a more aggressive phenotype. Lastly, 6 patients harbored estrogen receptor 1 (ESR1) mutations in cfDNA, suggesting resistance to endocrine therapy. Overall 9 of 42 patients (21%) had alterations in cfDNA that could herald a change in treatment. CONCLUSIONS: Targeted NGS of cfDNA has potential for monitoring response to targeted therapies through both mutations and gene amplification, for analysis of dynamic tumor heterogeneity and stratification to targeted therapy.