Search

Abstract Our study employs pooled CRISPR screens, integrating 2D and 3D culture models, to identify miRNAs critical in Breast Cancer (BC) tumoursphere formation. These screens combine with RNA-seq experiments allowing identification of miRNA signatures and targets essential for tumoursphere growth. miR-4787-3p exhibits significant up-regulation in BC, particularly in basal-like BCs, suggesting its association with aggressive disease. Surprisingly, despite its location within the 5’UTR of a protein coding gene, which defines DROSHA-independent transcription start site (TSS)-miRNAs, we find it dependant on both DROSHA and DICER1 for maturation. Inhibition of miR-4787-3p hinders tumoursphere formation, highlighting its potential as a therapeutic target in BC. Our study proposes elevated miR-4787-3p expression as a potential prognostic biomarker for adverse outcomes in BC. We find that protein-coding genes positively selected in the CRISPR screens are enriched of miR-4787-3p targets. Of these targets, we select ARHGAP17, FOXO3A, and PDCD4 as known tumour suppressors in cancer and experimentally validate the interaction of miR-4787-3p with their 3’UTRs. Our work illuminates the molecular mechanisms underpinning miR-4787-3p’s oncogenic role in BC. These findings advocate for clinical investigations targeting miR-4787-3p and underscore its prognostic significance, offering promising avenues for tailored therapeutic interventions and prognostic assessments in BC.

Abstract Background The interplay between exosomes and the tumor microenvironment (TME) remains unclear. We investigated the influence of exosomes on the TME in hepatocellular carcinoma (HCC), focusing on their mRNA expression profile. Methods mRNA expression profiles of exosomes were obtained from exoRBase. RNA sequencing data from HCC patients’ tumors were acquired from The Cancer Genome Atlas (TCGA) and the International Cancer Genome Consortium (ICGC). An exosome mRNA-related risk score model of prognostic value was established. The patients in the two databases were divided into high- and low-risk groups based on the median risk score value, and used to validate one another. Functional enrichment analysis was performed based on a differential gene prognosis model (DGPM). CIBERSORT was used to assess the abundance of immune cells in the TME. The correlation between the expression levels of immune checkpoint-related genes and DGPM was analyzed alongside the prediction value to drug sensitivity. Results A prognostic exosome mRNA-related 4-gene signature (DYNC1H1, PRKDC, CCDC88A, and ADAMTS5) was constructed and validated. A prognostic nomogram had prognostic ability for HCC. The genes for this model are involved in extracellular matrix, extracellular matrix (ECM)-receptor interaction, and the PI3K-Akt signaling pathway. Expression of genes here had a positive correlation with immune cell infiltration in the TME. Conclusions Our study results demonstrate that an exosome mRNA-related risk model can be established in HCC, highlighting the functional significance of the molecules in prognosis and risk stratification.

Abstract Introduction EXO-CD24 are exosomes genetically manipulated to over-express Cluster of Differentiation (CD) 24. It consists of two breakthrough technologies: CD24, the drug, as a novel immunomodulator that is smarter than steroids without any side effects, and exosomes as the ideal natural drug carrier. Methods A randomized, single blind, dose-finding phase IIb trial in hospitalized patients with mild to moderate Coronavirus disease 2019 (COVID-19) related Acute Respiratory Distress Syndrome (ARDS) was carried out in two medical centers in Athens. Patients received either 109 or 1010 exosome particles of EXO-CD24, daily, for five consecutive days and monitored for 28 days. Efficacy was assessed at day 7 among 91 patients who underwent randomization. The outcome was also compared in a post-hoc analysis with an income control group (n = 202) that fit the inclusion and exclusion criteria. Results The mean age was 49.4 (± 13.2) years and 74.4% were male. By day 7, 83.7% showed improved respiratory signs and 64% had better oxygen saturation (SpO2) (p

Abstract Invariant natural killer T (iNKT) cells, a unique T cell population, lend themselves for use as adoptive therapy due to diverse roles in orchestrating immune responses. Originally developed for use in cancer, agenT-797 is a donor-unrestricted allogeneic ex vivo expanded iNKT cell therapy. We conducted an open-label study in virally induced acute respiratory distress syndrome (ARDS) caused by the severe acute respiratory syndrome-2 virus (trial registration NCT04582201). Here we show that agenT-797 rescues exhausted T cells and rapidly activates both innate and adaptive immunity. In 21 ventilated patients including 5 individuals receiving veno-venous extracorporeal membrane oxygenation (VV-ECMO), there are no dose-limiting toxicities. We observe an anti-inflammatory systemic cytokine response and infused iNKT cells are persistent during follow-up, inducing only transient donor-specific antibodies. Clinical signals of associated survival and prevention of secondary infections are evident. Cellular therapy using off-the-shelf iNKT cells is safe, can be rapidly scaled and is associated with an anti-inflammatory response. The safety and therapeutic potential of iNKT cells across diseases including infections and cancer, warrants randomized-controlled trials.

The CCR/L5 axis is known for its role in immune regulation in a variety of settings and has been shown to have dichotomous functions in cancer, influencing both tumor progression and immune responses. Battaglin et al investigated its role using genomic and transcriptomic data from several datasets of patients with advanced colorectal cancer (CRC), including patients treated on CALGB/SWOG 80405, a trial of chemotherapy plus cetuximab versus bevacizumab, as well as a larger population of patients whose CRCs underwent commercially available Caris NGS and CODEai assays. These authors showed that CCR/L5 expression was both prognostic and predictive. They reported that low expression of the CCR/L5 axis was correlated with improved survival broadly, with particular benefit in patients treated with chemotherapy plus cetuximab. They demonstrated that high expression of CCR/L5 was associated with infiltration by negatively prognostic Tregs, M1 and M2 macrophages, myeloid-derived suppressor cells, and cancer-associated fibroblasts. They also showed that increased expression was correlated a wide variety of immune suppressive proteins, including PD-1, PD-L1, PD-L2, CTLA4, CD80, CD86, TIM3, IDO1, LAG3, and IFN-γ. This suggests mechanisms by which CRC resists anti-cancer immune responses. This study enhances our understanding of the role of the CCR/L5 axis in advanced CRC.

Abstract Differentiating between pancreatic ductal adenocarcinoma (PDAC) and cholangiocarcinoma (CCA) is crucial for the appropriate course of treatment, especially with advancements in the role of neoadjuvant chemotherapies for PDAC, compared to CCA. Furthermore, benign pancreaticobiliary diseases can mimic malignant disease, and indeterminate lesions may require repeated investigations to achieve a diagnosis. As bile flows in close proximity to these lesions, we aimed to establish a bile-based microRNA (miRNA) signature to discriminate between malignant and benign pancreaticobiliary diseases. We performed miRNA discovery by global profiling of 800 miRNAs using the NanoString nCounter platform in prospectively collected bile samples from malignant (n = 43) and benign (n = 14) pancreaticobiliary disease. Differentially expressed miRNAs were validated by RT-qPCR and further assessed in an independent validation cohort of bile from malignant (n = 37) and benign (n = 38) pancreaticobiliary disease. MiR-148a-3p was identified as a discriminatory marker that effectively distinguished malignant from benign pancreaticobiliary disease in the discovery cohort (AUC = 0.797 [95% CI 0.68–0.92]), the validation cohort (AUC = 0.772 [95% CI 0.66–0.88]), and in the combined cohorts (AUC = 0.752 [95% CI 0.67–0.84]). We also established a two-miRNA signature (miR-125b-5p and miR-194-5p) that distinguished PDAC from CCA (validation: AUC = 0.815 [95% CI 0.67–0.96]; and combined cohorts: AUC = 0.814 [95% CI 0.70–0.93]). Our research stands as the largest, multicentric, global profiling study of miRNAs in the bile from patients with pancreaticobiliary disease. We demonstrated their potential as clinically useful diagnostic tools for the detection and differentiation of malignant pancreaticobiliary disease. These bile miRNA biomarkers could be developed to complement current approaches for diagnosing pancreaticobiliary cancers. Graphical Abstract

Abstract Temozolomide (TMZ) represents the cornerstone of therapy for glioblastoma (GBM). However, acquisition of resistance limits its therapeutic potential. The human kinome is an undisputable source of druggable targets, still, current knowledge remains confined to a limited fraction of it, with a multitude of under‐investigated proteins yet to be characterized. Here, following a kinome‐wide RNAi screen, pantothenate kinase 4 (PANK4) isuncovered as a modulator of TMZ resistance in GBM. Validation of PANK4 across various TMZ‐resistant GBM cell models, patient‐derived GBM cell lines, tissue samples, as well as in vivo studies, corroborates the potential translational significance of these findings. Moreover, PANK4 expression is induced during TMZ treatment, and its expression is associated with a worse clinical outcome. Furthermore, a Tandem Mass Tag (TMT)‐based quantitative proteomic approach, reveals that PANK4 abrogation leads to a significant downregulation of a host of proteins with central roles in cellular detoxification and cellular response to oxidative stress. More specifically, as cells undergo genotoxic stress during TMZ exposure, PANK4 depletion represents a crucial event that can lead to accumulation of intracellular reactive oxygen species (ROS) and subsequent cell death. Collectively, a previously unreported role for PANK4 in mediating therapeutic resistance to TMZ in GBM is unveiled.

Abstract The accumulation of somatic mutations in healthy human tissues has been extensively characterized, but the mutational landscape of the healthy breast is still poorly understood. Our analysis of whole-genome sequencing shows that in line with other healthy organs, the healthy breast during the reproduction years accumulates mutations with age, with the rate of accumulation in the epithelium of 15.24 ± 5 mutations/year. Both epithelial and stromal compartments contain mutations in breast-specific driver genes, indicative of subsequent positive selection. Parity- and age-associated differences are evident in the mammary epithelium, partly explaining the observed difference in breast cancer risk amongst women of different childbearing age. Parity is associated with an age-dependent increase in the clone size of mutated epithelial cells, suggesting that older first-time mothers have a higher probability of accumulating oncogenic events in the epithelium compared to younger mothers or nulliparous women. In conclusion, we describe the reference genome of the healthy female human breast during reproductive years and provide evidence of how parity affects the genomic landscape of the mammary gland.

Abstract The PI3K/AKT/mTOR (PAM) signaling pathway is a highly conserved signal transduction network in eukaryotic cells that promotes cell survival, cell growth, and cell cycle progression. Growth factor signalling to transcription factors in the PAM axis is highly regulated by multiple cross-interactions with several other signaling pathways, and dysregulation of signal transduction can predispose to cancer development. The PAM axis is the most frequently activated signaling pathway in human cancer and is often implicated in resistance to anticancer therapies. Dysfunction of components of this pathway such as hyperactivity of PI3K, loss of function of PTEN, and gain-of-function of AKT, are notorious drivers of treatment resistance and disease progression in cancer. In this review we highlight the major dysregulations in the PAM signaling pathway in cancer, and discuss the results of PI3K, AKT and mTOR inhibitors as monotherapy and in co-administation with other antineoplastic agents in clinical trials as a strategy for overcoming treatment resistance. Finally, the major mechanisms of resistance to PAM signaling targeted therapies, including PAM signaling in immunology and immunotherapies are also discussed.

Background: Acquired resistance to targeted drugs is a major challenge in cancer. The drug-tolerant state has been proposed to be an initial step towards acquisition of real drug-resistance. Drug tolerant persister (DTP) cells are purported to survive during treatment and stay dormant for several years. Single cell sequencing can provide a comprehensive landscape of gene expression in DTP cells, which can facilitate investigation of heterogeneity of a drug tolerant state and identification of new anticancer targets. Methods: The genetic profiling of DTPs was explored by integrating Gene Expression Omnibus (GEO) datasets, and a prognostic signature of DTP-related genes (DTPRGs) in lung adenocarcinoma of TCGA LUAD cohort was constructed. The scores of infiltrating immune cells were calculated and activity of immune-related pathways was evaluated by single-sample gene set enrichment analysis (ssGSEA). Functional enrichment analysis of the DTPRGs between low- and high-risk groups was performed. Immune cell subtypes and immune-related pathways were analyzed. Results: An 11-gene panel (MT2A, UBE2S, CLTB, KRT7, IGFBP3, CTSH, NPC2, HMGA1, HNRNPAB, DTYMK, and IHNA) was established. DTPRGs were mainly correlated with nuclear division, chromosome segregation, and cell cycle pathways. Infiltration of immune cells was lower in the high-risk group while the inflammation-promoting and MCH-class I response pathway had higher activity in the high-risk group. A nomogram was generated with prognostic accuracy, further validated using clinical outcomes following therapy with epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs). Discussion: A prognostic model of lung adenocarcinoma based on DTPRGs was constructed. Targeting DTP cells is a potential therapeutic approach to prevent a drug tolerant state.

Abstract Purpose Pyroptosis, a type of programmed cell death, is implicated in the tumorigenesis, development and migration of cancer, which can be regulated by long non‐coding RNAs (lncRNAs). Our research aimed to investigate the prognostic role of pyroptosis‐related lncRNAs and the relationship to the tumor immune microenvironment through bioinformatics analysis. Methods The clinical and RNA‐sequencing data of bladder cancer patients were downloaded from The Cancer Genome Atlas (TCGA). And 412 bladder cancer subjects with clinical information were divided into training and testing cohort. And 52 reported pyroptosis‐related genes were used to screen pyroptosis‐related lncRNAs. A pyroptosis‐related lncRNA signature was constructed based on Cox regression analyses. Results A 9‐pyroptosis‐related‐lncRNA signature was identified to separate patients with bladder cancer into two groups. The prognosis of bladder cancer patients in the high‐risk group was significantly inferior compared with those in the low‐risk group. Risk scores were validated to develop an independent prognostic indicator based on multivariate Cox regression analysis. Receiver operating characteristic curve (ROC) analysis examined the signature on overall survival. The area under time‐dependent ROC curve (AUC) at 1‐, 3, and 5‐years measured 0.747, 0.783, and 0.768, respectively. Analysis of the immune landscape and PD‐L1 expression showed that PD‐L1 is upregulated in the high‐risk group. The immunocyte subtypes of the two groups were different. Conclusion A novel pyroptosis‐related lncRNA signature was identified with prognostic value for bladder cancer patients. Pyroptosis‐related lncRNAs have a potential role in cancer immunology and may serve as prognostic or therapeutic targets.

Abstract Background Radiation recall pneumonitis (RRP) is unpredictable but associated with severe radiation damage in previously irradiated fields. Chemotherapy and targeted drugs have been reported to contribute to RRP. Here we report a case of a patient with non-small cell lung cancer (NSCLC) who developed RRP following administration of immune checkpoint inhibitor (ICI) 18 months after the end of re-irradiation. Case presentation A 69-year-old man received adjuvant chemoradiotherapy post-operatively. He underwent thoracic re-irradiation for oligometastatic NSCLC. On second recurrence, pembrolizumab combined with nab-paclitaxel were administered. After six months, he developed symptoms of persistent cough and dyspnea, with consistent pneumonitis on CT images. The clinical time frame and significant radiographic evidence raised suspicion for RRP. Symptoms resolved after steroids. Conclusions RRP is a rare occurrence. Patients undergoing immunotherapy after prior irradiation may be at increased risk of this rare radiation pneumonitis.

Abstract A small but significant proportion of COVID‐19 patients develop life‐threatening cytokine storm. We have developed a new anti‐inflammatory drug, EXO‐CD24, a combination of an immune checkpoint (CD24) and a delivery platform (exosomes). CD24 inhibits the NF‐kB pathway and the production of cytokines/chemokines. EXO‐CD24 discriminates damage‐from pathogen‐associated molecular patterns (DAMPs and PAMPs) therefore does not interfere with viral clearance. EXO‐CD24 was produced and purified from CD24‐expressing 293‐TREx™ cells. Exosomes displaying murine CD24 (mCD24) were also created. EXO‐CD24/mCD24 were characterized and examined, for safety and efficacy, in vitro and in vivo. In a phase Ib/IIa study, 35 patients with moderate–high severity COVID‐19 were recruited and given escalating doses, 108–1010, of EXO‐CD24 by inhalation, QD, for 5 days. No adverse events related to the drug were observed up to 443–575 days. EXO‐CD24 effectively reduced inflammatory markers and cytokine/chemokine, although randomized studies are required. EXO‐CD24 may be a treatment strategy to suppress the hyper‐inflammatory response in the lungs of COVID‐19 patients and further serve as a therapeutic platform for other pulmonary and systemic diseases characterized by cytokine storm.

BackgroundEpidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) are effective treatments for non-small cell lung cancer (NSCLC) patients with activating EGFR mutations. There are many uncommon and rare mutations in the EGFR gene. The efficacy of the EGFR-TKIs is largely unknown for cancers harboring uncommon or rare EGFR mutations.Case PresentationA 69-year-old woman was diagnosed with adenocarcinoma cT4N2M1c, stage IVB. Next-generation sequencing (NGS) confirmed a rare EGFR V786M mutation. During chemotherapy, immune checkpoint inhibitor (ICI), and anti-angiogenic treatment, no radiological response was observed. Subsequent third-generation EGFR TKI showed a remarkable therapeutic effect. Structural prediction revealed that the V786M mutation induces conformational change at the dimer interface, without altering the ATP binding to the EGFR tyrosine kinase domain (TKD). Consistently, docking simulations indicated that the affinity of ATP to the V786M mutant was not disturbed, which explained the TKI sensitivity.ConclusionsOur data confirmed the activating role on EGFR V786M mutation. Together with structural predictions and clinical evidence for activity of TKIs against EGFR V786M mutations, these findings warrant further investigation.

Anaplastic lymphoma kinase (ALK) alterations in non-small cell lung cancer (NSCLC) can be effectively treated with a variety of ALK-targeted drugs. After the approval of the first-generation ALK inhibitor crizotinib which achieved better results in prolonging the progression-free survival (PFS) compared with chemotherapy, a number of next-generation ALK inhibitors have been developed including ceritinib, alectinib, brigatinib, and ensartinib. Recently, a potent, third-generation ALK inhibitor, lorlatinib, has been approved by the Food and Drug Administration (FDA) for the first-line treatment of ALK-positive (ALK+) NSCLC. These drugs have manageable toxicity profiles. Responses to ALK inhibitors are however often not durable, and acquired resistance can occur as on-target or off-target alterations. Studies are underway to explore the mechanisms of resistance and optimal treatment options beyond progression. Efforts have also been undertaken to develop further generations of ALK inhibitors. This review will summarize the current situation of targeting the ALK signaling pathway.

Recently, the oncogenic role of lemur tyrosine kinase 3 (LMTK3) has been well established in different tumor types, highlighting it as a viable therapeutic target. In the present study, using in vitro and cell-based assays coupled with biophysical analyses, we identify a highly selective small molecule LMTK3 inhibitor, namely C36. Biochemical/biophysical and cellular studies revealed that C36 displays a high in vitro selectivity profile and provides notable therapeutic effect when tested in the National Cancer Institute (NCI)-60 cancer cell line panel. We also report the binding affinity between LMTK3 and C36 as demonstrated via microscale thermophoresis (MST). In addition, C36 exhibits a mixed-type inhibition against LMTK3, consistent with the inhibitor overlapping with both the adenosine 5′-triphosphate (ATP)- and substrate-binding sites. Treatment of different breast cancer cell lines with C36 led to decreased proliferation and increased apoptosis, further reinforcing the prospective value of LMTK3 inhibitors for cancer therapy.

BackgroundAlternative splicing (AS) is a gene regulatory mechanism that drives protein diversity. Dysregulation of AS is thought to play an essential role in cancer initiation and development. This study aimed to construct a prognostic signature based on AS and explore the role in the tumor immune microenvironment (TIME) in lung adenocarcinoma.MethodsWe analyzed transcriptome profiling and clinical lung adenocarcinoma data from The Cancer Genome Atlas (TCGA) database and lists of AS-related and immune-related signatures from the SpliceSeq. Prognosis-related AS events were analyzed by univariate Cox regression analysis. Gene set enrichment analyses (GSEA) were performed for functional annotation. Prognostic signatures were identified and validated using univariate and multivariate Cox regression, LASSO regression, Kaplan–Meier survival analyses, and proportional hazards model. The context of TIME in lung adenocarcinoma was also analyzed. Gene and protein expression data of Cyclin-Dependent Kinase Inhibitor 2A (CDKN2A) were obtained from ONCOMINE and Human Protein Atlas. Splicing factor (SF) regulatory networks were visualized.ResultsA total of 19,054 survival-related AS events in lung adenocarcinoma were screened in 1,323 genes. Exon skip (ES) and mutually exclusive exons (ME) exhibited the most and fewest AS events, respectively. Based on AS subtypes, eight AS prognostic signatures were constructed. Patients with high-risk scores were associated with poor overall survival. A nomogram with good validity in prognostic prediction was generated. AUCs of risk scores at 1, 2, and 3 years were 0.775, 0.736, and 0.759, respectively. Furthermore, the prognostic signatures were significantly correlated with TIME diversity and immune checkpoint inhibitor (ICI)-related genes. Low-risk patients had a higher StromalScore, ImmuneScore, and ESTIMATEScore. AS-based risk score signature was positively associated with CD8+ T cells. CDKN2A was also found to be a prognostic factor in lung adenocarcinoma. Finally, potential functions of SFs were determined by regulatory networks.ConclusionTaken together, our findings show a clear association between AS and immune cell infiltration events and patient outcome, which could provide a basis for the identification of novel markers and therapeutic targets for lung adenocarcinoma. SF networks provide information of regulatory mechanisms.

BackgroundTargeted therapies have led to significant improvement in the management and prognosis of anaplastic lymphoma kinase (ALK)-positive non-small cell lung cancer (NSCLC). We performed a network meta-analysis of frontline treatment options of ALK-positive NSCLC to provide clinical guidance.MethodsPubMed, Embase, ClinicalTrials.gov, and international conference databases were searched to identify relevant trials from inception to June 30, 2021. Phase III randomized controlled trials (RCTs) comparing treatments for patients with ALK-positive advanced NSCLC in the first-line setting were included in a Bayesian network meta-analysis. Eligible studies reported at least one of the following clinical outcomes: progression-free survival (PFS), overall survival (OS), risk of the central nervous system (CNS) progression, adverse events (AEs) of grade (G) 3 or higher (G3 AEs), or serious AEs (SAEs). Hazard ratios (HRs) and CI for primary outcome of PFS and secondary outcome of OS and risk of CNS progression were obtained. A multivariate, consistency model, fixed-effects analysis was used in the network meta-analysis. Data on G3 AEs and SAEs were abstracted and meta-analyzed. Risk of bias (RoB) was assessed using the Cochrane Collaboration’s tool.ResultsNine RCTs comprising 2,484 patients were included with seven treatments: alectinib, brigatinib, ceritinib, crizotinib, ensartinib, lorlatinib, and chemotherapy. Compared with chemotherapy, ALK-tyrosine kinase inhibitors (TKIs) significantly prolong PFS and reduced risk of CNS progression except for ceritinib. Lorlatinib appears superior at reducing risk of CNS progression. None of the ALK-TKIs have a significantly prolonged OS as compared with chemotherapy. Lorlatinib increases the risk of G3 AEs as compared with alectinib (odds ratio 4.26 [95% CrI 1.22 to 15.53]), while alectinib caused the fewest G3 AEs.ConclusionsLorlatinib is associated with the highest PFS benefit and lowest risk of CNS progression benefits for patients with advanced ALK-positive NSCLC, compared with other first-line treatments, but with higher toxicity. The implementation of a newer generation of ALK-TKIs in the first-line treatment of ALK-positive NSCLC into current clinical practice is evolving rapidly.

An amendment to this paper has been published and can be accessed via the original article.

BackgroundSMARCA4, the essential ATPase subunit of SWI/SNF chromatin remodeling complex, regulates transcription through the control of chromatin structure and is increasingly thought to play significant roles in human cancers. This study aims to explore the potential role of SMARCA4 with a view to providing insights on pathologic mechanisms implicated here.MethodsThe potential roles of SMARCA4 in different tumors were explored based on The Cancer Genome Atlas (TCGA), Genotype-tissue expression (GTEx), Tumor Immune Estimation Resource (TIMER), and Gene Set Enrichment Analysis (GSEA) datasets. The expression difference, mutation and phosphorylation status, survival, pathological stage, DNA methylation, tumor mutation burden (TMB), microsatellite instability (MSI), mismatch repair (MMR), tumor microenvironment (TME), and immune cell infiltration related to SMARCA4 were analyzed.ResultsHigh expression levels of SMARCA4 were observed in most cancer types. SMARCA4 expression in tumor samples correlates with poor overall survival in several cancers. Lung adenocarcinoma cases with altered SMARCA4 showed a poorer prognosis. Enhanced phosphorylation levels of S613, S695, S699, and S1417 were observed in several tumors, including breast cancer. SMARCA4 correlated with tumor immunity and associated with different immune cells and genes in different cancer types. TMB, MSI, MMR, and DNA methylation correlated with SMARCA4 dysregulation in cancers. SMARCA4 expression was negatively associated with CD8+ T-cell infiltration in several tumors. Furthermore, the SWI/SNF superfamily-type complex and ATPase complex may be involved in the functional mechanisms of SMARCA4, albeit these data require further confirmation.ConclusionsOur study offers a comprehensive understanding of the oncogenic roles of SMARCA4 across different tumors. SMARCA4 may correlate with tumor immunity.

Radio-sensitizing nanoparticles are a potential method to increase the damage caused to cancerous cells during the course of radiotherapy. The distribution of these particles in a given targeted tumour is a relevant factor in determining the efficacy of nanoparticle-enhanced treatment. In this study, a three-part mathematical model is shown to predict the distribution of nanoparticles after direct injection into a tumour. In contrast with previous studies, here, a higher value of diffusivity for charged particles was used and the concentration profile of deposited particles was studied. Simulation results for particle concentrations both in the interstitial fluid and deposited onto cells are compared for different values of particle surface charges during and after injection. Our results show that particles with a negative surface charge can spread farther from the injection location as compared to uncharged particles with charged particles occupying 100% of the tumour volume compared to 8.8% for uncharged particles. This has implications for the future development of radiosensitizers and any associated trials.

BackgroundThe therapeutic efficacy of epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) in advanced EGFR-mutant lung squamous cell carcinoma (SCC) patients remains uncertain. Furthermore, the factors underlying the responsiveness have not been fully investigated. We therefore investigated the link between genomic profiles and EGFR-TKI efficacy.Material and MethodsWe consecutively enrolled stage IV, EGFR-mutant, and EGFR-TKI–treated patients with SCC. Patients with EGFR wild-type lung SCC and EGFR-mutant lung adenocarcinoma were consecutively enrolled as controls, and next-generation sequencing (NGS) was performed.ResultsIn total, 28 EGFR-mutant lung SCC, 41 EGFR-mutant lung adenocarcinoma, and 40 EGFR wild-type lung SCC patients were included. Among the patients with EGFR mutations, shorter progression-free survival (PFS) was observed in SCC compared to adenocarcinoma (4.6 vs. 11.0 months, P

BackgroundFirst-line treatment strategies for programmed death-ligand 1 (PD-L1) negative non-small cell lung cancer (NSCLC) patients include chemotherapy and combination with anti-angiogenesis drugs and/or immune checkpoint inhibitor. We conducted a Bayesian network meta-analysis to evaluate the efficacy of these therapeutic options.MethodsWe included phase III randomized controlled trials comparing two or more treatments in the first-line setting for NSCLC, including data in PD-L1–negative patients. First-line strategies were compared and ranked based on the effectiveness in terms of overall survival (OS) and progression-free survival (PFS). A rank was assigned to each treatment after Markov Chain Monte Carlo analyses.ResultsFourteen trials involving 14 regimens matched our eligibility criteria. For OS, none of the treatment were significantly more effective than chemotherapy. Nivolumab plus ipilimumab plus chemotherapy was probably the best option based on analysis of the treatment ranking (probability = 30.1%). For PFS, nivolumab plus chemotherapy plus bevacizumab, atezolizumab plus chemotherapy plus bevacizumab, and atezolizumab plus chemotherapy were statistically superior to chemotherapy in pairwise comparison. Nivolumab plus chemotherapy plus bevacizumab was likely to be the preferred option based on the analysis of the treatment ranking (probability = 72.9%).ConclusionsNivolumab plus chemotherapy, in combination with angiogenesis inhibition or anti-cytotoxic T-lymphocyte–associated antigen 4 (CTLA-4), had maximal benefits for NSCLC patient of PD-L1–negative expression. These findings may facilitate individualized treatment strategies. Safety at an individual patient level should be considered in decision making. Further validation is warranted.

During the current pandemic, the vast majority of COVID-19 patients experienced mild symptoms, but some had a potentially fatal aberrant hyperinflammatory immune reaction characterized by high levels of IL-6 and other cytokines. Modulation of this immune reaction has proven to be the only method of reducing mortality in severe and critical COVID-19. The anti-inflammatory drug baricitinib (Olumiant) has recently been strongly recommended by the WHO for use in COVID-19 patients because it reduces the risk of progressive disease and death. It is a Janus Kinase (JAK) 1/2 inhibitor approved for rheumatoid arthritis which was suggested in early 2020 as a treatment for COVID-19. In this review the AI-assisted identification of baricitinib, its antiviral and anti-inflammatory properties, and efficacy in clinical trials are discussed and compared with those of other immune modulators including glucocorticoids, IL-6 and IL-1 receptor blockers and other JAK inhibitors. Baricitinib inhibits both virus infection and cytokine signalling and is not only important for COVID-19 management but is “non-immunological”, and so should remain effective if new SARS-CoV-2 variants escape immune control. The repurposing of baricitinib is an example of how advanced artificial intelligence (AI) can quickly identify new drug candidates that have clinical benefit in previously unsuspected therapeutic areas.

Objects passed from one player to another have not been assessed for their ability to transmit severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We found that the surface of sport balls, notably a football, tennis ball, golf ball, and cricket ball could not harbour inactivated virus when it was swabbed onto the surface, even for 30 ​s. However, when high concentrations of 5000 ​dC/mL and 10,000 ​dC/mL are directly pipetted onto the balls, it could be detected after for short time periods. Sports objects can only harbour inactivated SARS-CoV-2 under specific, directly transferred conditions, but wiping with a dry tissue or moist ‘baby wipe’ or dropping and rolling the balls removes all detectable viral traces. This has helpful implications to sporting events.

Immune checkpoint inhibitors (ICIs) cause fewer toxicities than conventional chemotherapy. Although most of the immune-related adverse events (irAEs) are mild, reversible, and manageable, potentially severe and rare irAEs remain relevant. We present a 24-year-old man with advanced hereditary renal cancer who developed bilateral posterior uveitis and retinal detachment after systematic treatment of ICI and an anti-angiogenic drug. Axitinib and pembrolizumab were administered with a partial response and following the severe ocular irAE and systemic corticosteroid treatment was initiated. Our case indicates that ocular irAEs may occur rapidly. To the best of our knowledge, this is the first case of posterior uveitis and retinal detachment in hereditary renal cancer patients treated with ICI and anti-angiogenic drugs.

Abstract Baricitinib is an oral Janus kinase (JAK)1/JAK2 inhibitor approved for the treatment of rheumatoid arthritis (RA) that was independently predicted, using artificial intelligence (AI) algorithms, to be useful for COVID‐19 infection via proposed anti‐cytokine effects and as an inhibitor of host cell viral propagation. We evaluated the in vitro pharmacology of baricitinib across relevant leukocyte subpopulations coupled to its in vivo pharmacokinetics and showed it inhibited signaling of cytokines implicated in COVID‐19 infection. We validated the AI‐predicted biochemical inhibitory effects of baricitinib on human numb‐associated kinase (hNAK) members measuring nanomolar affinities for AAK1, BIKE, and GAK. Inhibition of NAKs led to reduced viral infectivity with baricitinib using human primary liver spheroids. These effects occurred at exposure levels seen clinically. In a case series of patients with bilateral COVID‐19 pneumonia, baricitinib treatment was associated with clinical and radiologic recovery, a rapid decline in SARS‐CoV‐2 viral load, inflammatory markers, and IL‐6 levels. Collectively, these data support further evaluation of the anti‐cytokine and anti‐viral activity of baricitinib and support its assessment in randomized trials in hospitalized COVID‐19 patients.

Background Subgroup analysis of clinical trials of PD-1/PD-L1 inhibitors have reported ethnic differences in outcomes. We systematically collected published data and performed a meta-analysis to compare therapeutic efficacy in Asian and non-Asian patients receiving PD-1/PD-L1 inhibitors. Methods Eligible studies included phase II and III prospective clinical trials with available subgroup data on Asian versus non-Asian populations. Overall survival (OS) and progression-free survival (PFS) were used to evaluate differences in outcome between Asian versus non-Asian cancer patients. Results A total of 11,020 cancer patients from 19 prospective randomized controlled clinical trials were included. The overall estimated HR for OS was 0.69 with 95% CI of 0.61–0.77 in Asian versus 0.82 with 95% CI of 0.77–0.88 in non-Asian patients. The estimated hazard ratio (HR) for PFS measured 0.54 (95% CI, 0.32–0.76) and 0.69 (95% CI, 0.54–0.85) in Asian and non-Asian patients, respectively. Pooled ratios of OS HRs and PFS HRs reported in Asian versus non-Asian cancer patients were 0.84 (95% CI, 0.75–0.94) and 0.78 (95% CI, 0.59–0.97), respectively. Conclusions This meta-analysis shows for the first time that Asian cancer patients have a significantly improved survival benefit than non-Asian patients receiving PD-1/PD-L1 inhibitor-based therapy.

Abstract Glioblastoma (GBM) is the most aggressive type of primary brain tumours. Anti-angiogenic therapies (AAT), such as bevacizumab, have been developed to target the tumour blood supply. However, GBM presents mechanisms of escape from AAT activity, including a speculated direct effect of AAT on GBM cells. Furthermore, bevacizumab can alter the intercellular communication of GBM cells with their direct microenvironment. Extracellular vesicles (EVs) have been recently described as main acts in the GBM microenvironment, allowing tumour and stromal cells to exchange genetic and proteomic material. Herein, we examined and described the alterations in the EVs produced by GBM cells following bevacizumab treatment. Interestingly, bevacizumab that is able to neutralise GBM cells-derived VEGF-A, was found to be directly captured by GBM cells and eventually sorted at the surface of the respective EVs. We also identified early endosomes as potential pathways involved in the bevacizumab internalisation by GBM cells. Via MS analysis, we observed that treatment with bevacizumab induces changes in the EVs proteomic content, which are associated with tumour progression and therapeutic resistance. Accordingly, inhibition of EVs production by GBM cells improved the anti-tumour effect of bevacizumab. Together, this data suggests of a potential new mechanism of GBM escape from bevacizumab activity.

In pancreatic ductal adenocarcinoma, TGF-β/Activin induce epithelial-to-mesenchymal transition (EMT) and stemness. Here, the authors show that TGF-β induces pro-tumourigenic miR-100 and miR-125b, but blocks anti-tumourigenic let-7a maturation via LIN28B, regulating pathways to promote stemness, EMT and tumourigenesis.

Five days following the 3rd cycle of nivolumab, a monoclonal antibody, which acts as immune checkpoint inhibitor against the programmed cell death protein-1, for metastatic lung adenocarcinoma, a 56-year-old woman presented at the hospital critically ill. On admission, she had severe diabetic ketoacidosis (DKA), as evidenced by venous glucose of 47 mmol/L, blood ketones of 7.5 mmol/L, pH of 6.95 and bicarbonate of 6.6 mmol/L. She has had no personal or family history of diabetes mellitus (DM), while random venous glucose, measured 1 week prior to hospitalisation, was 6.1 mmol/L. On admission, her HbA1c was 8.2% and anti-GAD antibodies were 12 kIU/L (0–5 kU/L), while islet cell antibodies and serum C-peptide were undetectable. Nivolumab was recommenced without the development of other immune-mediated phenomena until 6 months later, when she developed hypothyroidism with TSH 18 U/L and low free T4. She remains insulin dependent and has required levothyroxine replacement, while she has maintained good radiological and clinical response to immunotherapy. This case is notable for the rapidity of onset and profound nature of DKA at presentation, which occurred two months following commencement of immunotherapy. Despite the association of nivolumab with immune-mediated endocrinopathies, only a very small number of patients developing type 1 DM has been reported to date. Patients should be closely monitored for hyperglycaemia and thyroid dysfunction prior to and periodically during immunotherapy.

In human leukemia, lineage-specific genes represent predominant targets of deletion, with lymphoid-specific genes frequently affected in lymphoid leukemia and myeloid-specific genes in myeloid leukemia. To investigate the basis of lineage-specific alterations, we analyzed global DNA damage in primary B cell precursors expressing leukemia-inducing oncogenes by ChIP-seq. We identified more than 1,000 sensitive regions, of which B lineage-specific genes constitute the most prominent targets. Identified hotspots at B lineage genes relate to DNA-DSBs, affect genes that harbor genomic lesions in human leukemia, and associate with ectopic deletion in successfully transformed cells. Furthermore, we show that most identified regions overlap with gene bodies of highly expressed genes and that induction of a myeloid lineage phenotype in transformed B cell precursors promotes de novo DNA damage at myeloid loci. Hence, we demonstrate that lineage-specific transcription predisposes lineage-specific genes in transformed B cell precursors to DNA damage, which is likely to promote the frequent alteration of lineage-specific genes in human leukemia.

The importance and role of the estrogen receptor (ER) pathway has been well-documented in both breast cancer (BC) development and progression. The treatment of choice in women with metastatic breast cancer (MBC) is classically divided into a variety of endocrine therapies, 3 of the most common being: selective estrogen receptor modulators (SERM), aromatase inhibitors (AI) and selective estrogen receptor down-regulators (SERD). In a proportion of patients, resistance develops to endocrine therapy due to a sophisticated and at times redundant interference, at the molecular level between the ER and growth factor. The progression to endocrine resistance is considered to be a gradual, step-wise process. Several mechanisms have been proposed but thus far none of them can be defined as the complete explanation behind the phenomenon of endocrine resistance. Although multiple cellular, molecular and immune mechanisms have been and are being extensively studied, their individual roles are often poorly understood. In this review, we summarize current progress in our understanding of ER biology and the molecular mechanisms that predispose and determine endocrine resistance in breast cancer patients.

Purpose: To investigate the impact of programmed death-ligand 1 (PD-L1) expression, oncogenic mutations, and clinical characteristics on survival after treatment with anti-PD-1/PD-L1 antibodies versus chemotherapy in non-small cell lung cancer (NSCLC). Patients and Methods: This meta-analysis included randomized trials comparing anti-PD-1/PD-L1 antibodies with chemotherapy. Hazard ratios (HRs) and 95% confidence interval (CI) for overall survival (OS) for the trial population and prespecified subgroups were extracted. We calculated pooled estimates of treatment efficacy using the fixed-effects or random-effects model when appropriate. All statistical tests were two sided. Results: Seven trials involving 3871 patients were included. The pooled results showed that anti-PD-1/PD-L1 immunotherapy significantly prolonged OS (HR: 0.73; 95% CI, 0.63 to 0.84) and PFS (HR: 0.84; 95% CI, 0.71 to 0.99) compared to chemotherapy. OS benefit from immunotherapy were observed in all PD-L1 expression subgroups (negative: HR, 0.79; 95% CI, 0.67 to 0.93; weak-positive: HR, 0.80; 95% CI, 0.67 to 0.95; strong-positive: HR, 0.61; 95% CI, 0.47 to 0.78). Strong-positive PD-L1 expression showed a trend towards more benefit compared to weak-positive PD-L1 expression (interaction P = 0.08). KRAS mutant (HR: 0.60; 95% CI, 0.39 to 0.93), EGFR wild-type (HR: 0.73; 95% CI, 0.61 to 0.87) and smoker (HR: 0.70; 95% CI, 0.60 to 0.83) subgroups achieved significant OS benefit from immunotherapy compared to corresponding subgroups. Survival benefit to immunotherapy was not significantly associated with histology, CNS metastases, age, gender and performance status. Conclusion: This study confirmed that treatment with anti-PD-1/PD-L1 improves overall survival compared with chemotherapy. Benefit was seen, regardless of PD-L1 expression levels; however, PD-L1 strong-positive patients trended to have greatest benefit. Patients with a KRAS mutant or EGFR wild-type tumor have improved survival benefit from immunotherapy compared with KRAS wild-type or EGFR mutant NSCLC, respectively.

Tyrosine kinases (TKs) play an essential role in regulating various cellular activities and dysregulation of TK signaling contributes to oncogenesis. However, less than half of the TKs have been thoroughly studied. Through a combined use of RNAi and stable isotope labeling with amino acids in cell culture (SILAC)-based quantitative proteomics, a global functional proteomic landscape of TKs in breast cancer was recently revealed highlighting a comprehensive and highly integrated signaling network regulated by TKs (Stebbing et al., 2015) [1]. We collate the enormous amount of the proteomic data in an open access platform, providing a valuable resource for studying the function of TKs in cancer and benefiting the science community. Here we present a detailed description related to this study (Stebbing et al., 2015) [1] and the raw data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the identifier http://www.ebi.ac.uk/pride/archive/projects/PXD002065. Keywords: Breast cancer, Cell signaling, Proteomics, SILAC, Tyrosine kinases

Pulmonary aspergillosis is an opportunistic fungal infection affecting immunocompromised individuals. Increasing understanding of natural killer (NK) cell immunobiology has aroused considerable interest around the role of NK cells in pulmonary aspergillosis in the immunocompromised host. Murine studies indicate that NK cells play a critical role in pulmonary clearance of A. fumigatus. We show that the in vitro interaction between NK cells and A. fumigatus induces partial activation of NK cell immune response, characterised by low-level production of IFN-γ, TNF-α, MIP-1α, MIP-1β, and RANTES, polarisation of lytic granules and release of fungal DNA. We observed a contact-dependent down-regulation of activatory receptors NKG2D and NKp46 on the NK cell surface, and a failure of full granule release. Furthermore, the NK cell cytokine-mediated response to leukaemic cells was impaired in the presence of A. fumigatus. These observations suggest that A. fumigatus-mediated NK cell immunoparesis may represent an important mechanism of immune evasion during pulmonary aspergillosis.

Next generation sequencing (NGS) is becoming increasingly integrated into oncological practice and clinical research. NGS methods have also provided evidence for clonal evolution of cancers during disease progression and treatment. The number of variants associated with response to specific therapeutic agents keeps increasing. However, the identification of novel driver mutations as opposed to passenger (phenotypically silent or clinically irrelevant) mutations remains a major challenge. We conducted targeted exome sequencing of advanced solid tumors from 44 pre-treated patients with solid tumors including breast, colorectal and lung carcinomas, neuroendocrine tumors, sarcomas and others. We catalogued established driver mutations and putative new drivers as predicted by two distinct algorithms. The established drivers we detected were consistent with published observations. However, we also detected a significant number of mutations with driver potential never described before in each tumor type we studied. These putative drivers belong to key cell fate regulatory networks, including potentially druggable pathways. Should our observations be confirmed, they would support the hypothesis that new driver mutations are selected by treatment in clinically aggressive tumors, and indicate a need for longitudinal genomic testing of solid tumors to inform second line cancer treatment.

LMTK3 is an oncogenic receptor tyrosine kinase (RTK) implicated in various types of cancer, including breast, lung, gastric, and colorectal cancer. It is localized in different cellular compartments, but its nuclear function has not been investigated so far. We mapped LMTK3 binding across the genome using ChIP-seq and found that LMTK3 binding events are correlated with repressive chromatin markers. We further identified KRAB-associated protein 1 (KAP1) as a binding partner of LMTK3. The LMTK3/KAP1 interaction is stabilized by PP1α, which suppresses KAP1 phosphorylation specifically at LMTK3-associated chromatin regions, inducing chromatin condensation and resulting in transcriptional repression of LMTK3-bound tumor suppressor-like genes. Furthermore, LMTK3 functions at distal regions in tethering the chromatin to the nuclear periphery, resulting in H3K9me3 modification and gene silencing. In summary, we propose a model where a scaffolding function of nuclear LMTK3 promotes cancer progression through chromatin remodeling.

[This corrects the article DOI: 10.1371/journal.pone.0085935.].

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

BACKGROUND:Intratumoral heterogeneity may help drive resistance to targeted therapies in cancer. In breast cancer, the presence of nodal metastases is a key indicator of poorer overall survival. The aim of this study was to identify somatic genetic alterations in early dissemination of breast cancer by whole genome next generation sequencing (NGS) of a primary breast tumor, a matched locally-involved axillary lymph node and healthy normal DNA from blood. METHODS:Whole genome NGS was performed on 12 µg (range 11.1-13.3 µg) of DNA isolated from fresh-frozen primary breast tumor, axillary lymph node and peripheral blood following the DNA nanoball sequencing protocol. Single nucleotide variants, insertions, deletions, and substitutions were identified through a bioinformatic pipeline and compared to CIN25, a key set of genes associated with tumor metastasis. RESULTS:Whole genome sequencing revealed overlapping variants between the tumor and node, but also variants that were unique to each. Novel mutations unique to the node included those found in two CIN25 targets, TGIF2 and CCNB2, which are related to transcription cyclin activity and chromosomal stability, respectively, and a unique frameshift in PDS5B, which is required for accurate sister chromatid segregation during cell division. We also identified dominant clonal variants that progressed from tumor to node, including SNVs in TP53 and ARAP3, which mediates rearrangements to the cytoskeleton and cell shape, and an insertion in TOP2A, the expression of which is significantly associated with tumor proliferation and can segregate breast cancers by outcome. CONCLUSION:This case study provides preliminary evidence that primary tumor and early nodal metastasis have largely overlapping somatic genetic alterations. There were very few mutations unique to the involved node. However, significant conclusions regarding early dissemination needs analysis of a larger number of patient samples.

LC-MS/MS phospho-proteomics is an essential technology to help unravel the complex molecular events that lead to and propagate cancer. We have developed a global phospho-proteomic workflow to determine activity of signaling pathways and drug targets in pancreatic cancer tissue for clinical application.Peptides resulting from tryptic digestion of proteins extracted from frozen tissue of pancreatic ductal adenocarcinoma and background pancreas (n = 12), were labelled with tandem mass tags (TMT 8-plex), separated by strong cation exchange chromatography, then were analysed by LC-MS/MS directly or first enriched for phosphopeptides using IMAC and TiO2, prior to analysis. In-house, commercial and freeware bioinformatic platforms were used to identify relevant biological events from the complex dataset.Of 2,101 proteins identified, 152 demonstrated significant difference in abundance between tumor and non-tumor tissue. They included proteins that are known to be up-regulated in pancreatic cancer (e.g. Mucin-1), but the majority were new candidate markers such as HIPK1 & MLCK. Of the 6,543 unique phosphopeptides identified (6,284 unique phosphorylation sites), 635 showed significant regulation, particularly those from proteins involved in cell migration (Rho guanine nucleotide exchange factors & MRCKα) and formation of focal adhesions. Activator phosphorylation sites on FYN, AKT1, ERK2, HDAC1 and other drug targets were found to be highly modulated (≥2 fold) in different cases highlighting their predictive power.Here we provided critical information enabling us to identify the common and unique molecular events likely contributing to cancer in each case. Such information may be used to help predict more bespoke therapy suitable for an individual case.

Genes involved in the carcinogenetic mechanisms underlying malignant pleural mesothelioma (MPM) are still poorly characterized. So far, mesothelin (MSLN) has aroused the most interest. It encodes for a membrane glycoprotein, frequently over-expressed in various malignancies such as MPM, and ovarian and pancreatic cancers. It has been proposed as a diagnostic and immunotherapeutic target with promising results. However, an alternative therapeutic approach seems to rise, whereby synthetic molecules, such as antisense oligonucleotides, could be used to inhibit MSLN activity. To date, such a gene-level inhibition has been attempted in two studies only, both on pancreatic and ovarian carcinoma cell lines, with the use of silencing RNA approaches. With regard to MPM, only one cell line (H2373) has been employed to study the effects of MSLN depletion. Indeed, the knowledge on the role of MSLN in MPM needs expanding. Accordingly, we investigated the expression of MSLN in a panel of three MPM cell lines, i.e., NCI-H28, Mero-14, and IstMes2; one non-MPM cell line was used as reference (Met5A). MSLN knock-down experiments on MSLN-overexpressing cells were also performed through silencing RNA (siRNA) to verify whether previous findings could be generalized to a different set of cell cultures. In agreement with previous studies, transient MSLN-silencing caused decreased proliferation rate and reduced invasive capacity and sphere formation in MSLN-overexpressing Mero-14 cells. Moreover, MSLN-siRNA combined with cisplatin, triggered a marked increase in apoptosis and a decrease in proliferation as compared to cells treated with each agent alone, thereby suggesting a sensitizing effect of siRNA towards cisplatin. In summary, our findings confirm that MSLN should be considered a key molecular target for novel gene-based targeted therapies of cancer.

The growth arrest-specific transcript 5 gene (GAS5) encodes a long noncoding RNA (lncRNA) and hosts a number of small nucleolar RNAs (snoRNAs) that have recently been implicated in multiple cellular processes and cancer. Here, we investigate the relationship between DNA damage, p53, and the GAS5 snoRNAs to gain further insight into the potential role of this locus in cell survival and oncogenesis both in vivo and in vitro.We used quantitative techniques to analyse the effect of DNA damage on GAS5 snoRNA expression and to assess the relationship between p53 and the GAS5 snoRNAs in cancer cell lines and in normal, pre-malignant, and malignant human colorectal tissue and used biological techniques to suggest potential roles for these snoRNAs in the DNA damage response.GAS5-derived snoRNA expression was induced by DNA damage in a p53-dependent manner in colorectal cancer cell lines and their levels were not affected by DICER. Furthermore, p53 levels strongly correlated with GAS5-derived snoRNA expression in colorectal tissue.In aggregate, these data suggest that the GAS5-derived snoRNAs are under control of p53 and that they have an important role in mediating the p53 response to DNA damage, which may not relate to their function in the ribosome. We suggest that these snoRNAs are not processed by DICER to form smaller snoRNA-derived RNAs with microRNA (miRNA)-like functions, but their precise role requires further evaluation. Furthermore, since GAS5 host snoRNAs are often used as endogenous controls in qPCR quantifications we show that their use as housekeeping genes in DNA damage experiments can lead to inaccurate results.

BackgroundAnalysis of circulating tumor cells (CTCs) provides real-time measures of cancer sub-populations with potential for CTC-directed therapeutics. We examined whether lapatinib which binds both HER2 and EGFR could induce depletion of the EGFR-positive pool of CTCs, which may in turn lead to clinical benefits.Patients and methodsPatients with metastatic breast cancer and HER2 non-amplified primary tumors with EGFR-positive CTCs were recruited and lapatinib 1500 mg daily was administered, in a standard two step phase 2 trial.ResultsThere were no responses leading to termination at the first analysis with 16 patients recruited out of 43 screened. In 6 out of 14 (43%) individuals eligible for the efficacy analysis, a decrease in CTCs was observed with most of these having a greater decrease in their EGFR-positive CTC pool.ConclusionsThis is one of the first studies of CTC-directed therapeutics and suggests that lapatinib monotherapy is not having any demonstrable clinical effects by reducing the EGFR-positive pool of CTCs in HER2 non-amplified primary tumors. Our attempt to expand the pool of patients eligible for a targeted therapy was unsuccessful; the role of clonal populations in cancer biology and therapeutic strategies to control them will require extensive evaluation in years to come.Trial registrationClinical trials.gov NCT00820924.