Dynamic monitoring of cerebrospinal fluid circulating tumor DNA to identify unique genetic profiles of brain metastatic tumors and better predict intracranial tumor responses in non-small cell lung cancer patients with brain metastases: a prospective cohort study (GASTO 1028).

BACKGROUND: Due to the blood-brain barrier, plasma is not an ideal source to evaluate the genetic characteristics of central nervous system tumors. Thus, cerebrospinal fluid (CSF) is becoming an alternative biopsy type to evaluate the genetic landscape of intracranial tumors. We aimed to explore the genetic profiles of CSF-derived circulating tumor DNA (ctDNA) to predict intracranial tumor responses and monitor mutational evolution during the treatment of non-small cell lung cancer (NSCLC) patients with brain metastases. METHODS: We conducted a prospective study of 92 newly diagnosed NSCLC patients with brain metastases. Paired CSF and plasma samples were collected at baseline, 8 weeks after treatment initiation, and disease progression. All samples underwent next-generation sequencing of 425 cancer-related genes. RESULTS: At baseline, the positive detection rates of ctDNA in CSF, plasma, and extracranial tumors were 63.7% (58/91), 91.1% (82/90), and 100% (58/58), respectively. A high level of genetic heterogeneity was observed between paired CSF and plasma, while concordance in driver mutations was also observed. A higher number of unique copy number variations was detected in CSF-ctDNA than in plasma. ctDNA positivity of CSF samples at baseline was associated with poor outcomes (HR=2.565, P=0.003). Moreover, patients with ≥ 50% reductions in the concentrations of CSF ctDNA after 8 weeks of treatment had significantly longer intracranial progression-free survivals (PFS) than patients with < 50% reductions in CSF ctDNA concentrations (13.27 months vs 6.13 months, HR=0.308, P=0.017). A ≥ 50% reduction in CSF ctDNA concentrations had better concordance with radiographic intracranial tumor responses than plasma. A ≥ 50% reduction in plasma ctDNA concentrations was also associated with longer extracranial PFS (11.57 months vs 6.20 months, HR=0.406, P=0.033). Based on clonal evolution analyses, the accumulation of subclonal mutations in CSF ctDNA was observed after 8 weeks of treatment. The clonal mutations that remained in more than 80% in CSF after 8 weeks also predicted shorter intracranial PFS (HR=3.785, P=0.039). CONCLUSIONS: CSF ctDNA exhibited unique genetic profiles of brain metastases, and dynamic changes in CSF ctDNA could better predict intracranial tumor responses and track clonal evolution during treatment in NSCLC patients with brain metastases. TRIAL REGISTRATION: ClinicalTrials.gov identifier: NCT03257735.

N6-methyladenosine induced miR-143-3p promotes the brain metastasis of lung cancer via regulation of VASH1.

BACKGROUND: Brain metastasis (BM) is one of the principal causes of mortality for lung cancer patients. While the molecular events that govern BM of lung cancer remain frustrating cloudy. METHODS: The miRNA expression profiles are checked in the paired human BM and primary lung cancer tissues. The effect of miR-143-3p on BM of lung cancer cells and its related mechanisms are investigated. RESULTS: miR-143-3p is upregulated in the paired BM tissues as compared with that in primary cancer tissues. It can increase the invasion capability of in vitro blood brain barrier (BBB) model and angiogenesis of lung cancer by targeting the three binding sites of 3'UTR of vasohibin-1 (VASH1) to inhibit its expression. Mechanistically, VASH1 can increase the ubiquitylation of VEGFA to trigger the proteasome mediated degradation, further, it can endow the tubulin depolymerization through detyrosination to increase the cell motility. m6A methyltransferase Mettl3 can increase the splicing of precursor miR-143-3p to facilitate its biogenesis. Moreover, miR-143-3p/VASH1 axis acts as adverse prognosis factors for in vivo progression and overall survival (OS) rate of lung cancer. CONCLUSIONS: Our work implicates a causal role of the miR-143-3p/VASH1 axis in BM of lung cancers and suggests their critical roles in lung cancer pathogenesis.

Genes associated with increased brain metastasis risk in non-small cell lung cancer: Comprehensive genomic profiling of 61 resected brain metastases versus primary non-small cell lung cancer (Guangdong Association Study of Thoracic Oncology 1036).

BACKGROUND: Patients with brain metastases (BMs) have a poor prognosis and limited therapeutic options. Lung cancer is the most common primary malignancy giving rise to BMs; thus, understanding the molecular mechanisms behind increased BM risk is essential for identifying therapeutic targets and developing effective interventions. METHODS: Sixty-one patients who underwent surgical resection of primary non-small cell lung cancer (NSCLC) and BMs were retrospectively studied. Comprehensive genomic profiling of primary NSCLC and matched BMs was performed with next-generation sequencing targeting 416 cancer-relevant genes. RESULTS: Mutations of major drivers, including EGFR, KRAS, TP53, and ALK, were highly concordant between primary NSCLC and matched BMs (>80%), whereas discordance suggested the unique genomic evolution and oncogenic mechanisms of NSCLC BMs. BMs also demonstrated higher levels of copy number variations in comparison with primary NSCLC. Furthermore, the alterations of genes encoding CDK4/CCND1, CDKN2A/2B, and PI3K signaling pathways were enriched in BMs, and this suggested their correlation with increased metastatic risk. Indeed, patients with activated PI3K signaling in their primary NSCLC had significantly shorter BM-free survival (hazard ratio, 8.49; P = .0005). In addition, mutated TP53 or an activated WNT pathway via CTNNB1, APC, and AXIN2 mutations trended toward shorter BM-free intervals but not significantly so. CONCLUSIONS: These findings yield detailed insights into the genomic complexity and heterogeneity of primary NSCLC and matched BMs. This study highlights the significant correlation of PI3K signaling with increased metastatic risk in patients with NSCLC and identifies genomic alterations enriched in NSCLC BMs that could serve as prognostic markers and potential therapeutic targets for treating patients with NSCLC BMs.

The mechanism of lncRNAs in the crosstalk between epithelial-mesenchymal transition and tumor microenvironment for early colon adenocarcinoma based on molecular subtyping.

A large number of colon adenocarcinoma (COAD) patients are already advanced when diagnosed. In this study, we aimed to further understand the mechanism of tumor development in early COAD by focusing on epithelial-mesenchymal transition (EMT) and long non-coding RNAs (lncRNAs). Expression profiles of early COAD patients were obtained from public databases. EMT-related lncRNAs were used as a basis for constructing molecular subtypes through unsupervised consensus clustering. Genomic features, pathways and tumor microenvironment (TME) were compared between two subtypes. LncATLAS database was applied to analyze the relation between lncRNAs and transcription factors (TFs). First order partial correlation analysis was conducted to identify key EMT-related lncRNAs.C1 and C2 subtypes with distinct prognosis were constructed. Oncogenic pathways such as EMT, KRAS signaling, JAK-STAT signaling, and TGF-ß signaling were significantly enriched in C2 subtype. Higher immune infiltration and expression of immune checkpoints were also observed in C2 subtype, suggesting the key EMT-related lncRNAs may play a critical role in the modulation of TME. In addition, JAK-STAT signaling pathway was obviously enriched in upregulated TFs in C2 subtype, which indicated a link between key lncRNAs and JAK-STAT signaling that may regulate TME. The study further expanded the research on the role of EMT-related lncRNAs in the early COAD. The six identified EMT-related lncRNAs could serve as biomarkers for early screening COAD.

Impact of STAT1 polymorphisms on crizotinib-induced hepatotoxicity in ALK-positive non-small cell lung cancer patients.

PURPOSE: Crizotinib is the first-line small molecule tyrosine kinase inhibitor for ALK-positive non-small cell lung cancer. In this study, a retrospective pharmacogenomics investigation was conducted to explore the relationship between genes related to RTK downstream signaling pathways and crizotinib-induced hepatic toxicity in ALK-positive NSCLC patients. METHODS: The variable importance analysis of random forest algorithm was applied to identify the significant features which contribute to the crizotinib sensitivity in Cancer Cell Line Encyclopedia (CCLE) database. The KEGG and reactome pathway enrichment analysis were conducted with EnrichR. The differential expression genes were identified with R package DESeq2 in CCLE liver derived cell lines between crizotinib sensitive and resistant groups. From 2012 to 2015, 42 NSCLC patients were enrolled in this study. 90 polymorphisms were genotyped using the Sequenom Massarray system. Sequencing of HGFR (c-Met) genes was carried out on the Ion Torrent Proton. RESULTS: In total, 66.7% NSCLC patients suffered from crizotinib-induced liver toxicity and 11.9% progressed to severe hepatic toxicity. The features with the top importance from classification and regression random forest model were enriched in RTK downstream signaling pathways (JAK/STAT, RAS/RAF/MAPK, PI3K/AKT pathways) and immune system-related pathways. Collagen family genes (COL1A1, COL1A2, COL6A1, COL5A1) and other extracellular matrix protein (TNC, TAGLN, TENM2, EDIL3, VCAN, CNN1, SH3BP4, TAGLN), which were closely related to MAPK-ERK signaling pathways, were significantly enriched in crizotinib resistant cell lines. In multiple logistic regression, STAT1 rs10208033 (T > C) was significantly associated with crizotinib-induced liver toxicity (OR = 6.733, 95% CI 1.406-32.24, P = 0.017). Compared with non-CC, OR is 5.5 (95% CI 1.219-24.81, P = 0.027) for STAT1 rs10208033 CC genotype to develop crizotinib-induced liver toxicity. Further cell viability test in human fetal hepatocyte line, L-02, reveals that the STAT1 inhibitor might protect hepatocyte cells from the toxicity caused by crizotinib. CONCLUSION: Polymorphism of rs10208033 is a potential biomarker for predicting crizotinib-induced hepatotoxicity. These results suggest that STAT1 plays an important role in crizotinib-induced hepatotoxicity. Further studies are needed to confirm our finding and understand the underlying mechanisms.

Management of Non-Small-Cell Lung Cancer Patients Initially Diagnosed With 1 to 3 Synchronous Brain-Only Metastases: A Retrospective Study.

BACKGROUND: The treatment options for newly diagnosed non-small-cell lung cancer (NSCLC) patients with 1 to 3 synchronous brain metastases (BM) remain controversial. The current study aimed to comprehensively analyze the characteristics, local treatment paradigms, and survival outcomes in these populations. PATIENTS AND METHODS: A total of 252 NSCLC patients initially diagnosed with 1 to 3 synchronous brain-only metastases were enrolled onto this study. Local therapy (LT) to primary lung tumors (PLT) and BM included surgery, radiotherapy, or both. Median overall survival (mOS) was measured among patients who received LT to both PLT and BM (all-LT group), patients who were treated with LT to either PLT or BM (part-LT group), and patients who did not receive any LT (non-LT group). RESULTS: The mOS for all-LT (n = 70), part-LT (n = 113), and non-LT (n = 69) groups was 33.2, 18.5, and 16.8 months, respectively (P = .002). The OS rates at 5 years for the all-LT, part-LT, and non-LT groups were 25.5%, 16.2%, and 0, respectively. Multivariable analysis revealed that all-LT versus non-LT, pretreatment Karnofsky performance status > 70, histology of adenocarcinoma, thoracic stage I-II, EGFR mutation, ALK positive, and second-line systemic therapies were independent prognostic factors for improved mOS. CONCLUSIONS: The current study showed that LT for both PLT and BM is associated with superior OS in appropriately selected NSCLC patients initially diagnosed with 1 to 3 synchronous BM. Prospective trials are urgently needed to confirm this finding.

Reversal of ABCB1-related multidrug resistance by ERK5-IN-1.

BACKGROUND: Inhibition of ABC transporters is considered the most effective way to circumvent multidrug resistance (MDR). In the present study, we evaluated the MDR modulatory potential of ERK5-IN-1, a potent extracelluar signal regulated kinase 5 (ERK5) inhibitor. METHODS: The cytotoxicity and MDR reversal effect of ERK5-IN-1 were assessed by MTT assay. The KBv200-inoculated nude mice xenograft model was used for the in vivo study. Doxorubicin efflux and accumulation were measured by flow cytometry. The modulation of ABCB1 activity was measured by colorimetric ATPase assay and [125I]-iodoarylazidoprazosin (IAAP) photolabeling assay. Effect of ERK5-IN-1 on expression of ABCB1 and its downstream markers was measured by PCR and/or Western blot. Cell surface expression and subcellular localization of ABCB1 were tested by flow cytometry and immunofluorescence. RESULTS: Our results showed that ERK5-IN-1 significantly increased the sensitivity of vincristine, paclitaxel and doxorubicin in KBv200, MCF7/adr and HEK293/ABCB1 cells, respectively. This effect was not found in respective drug sensitive parental cell lines. Moreover, in vivo combination studies showed that ERK5-IN-1 effectively enhanced the antitumor activity of paclitaxel in KBv200 xenografts without causing addition toxicity. Mechanistically, ERK5-IN-1 increased intracellular accumulation of doxorubicin dose dependently by directly inhibiting the efflux function of ABCB1. ERK5-IN-1 stimulated the ABCB1 ATPase activity and inhibited the incorporation of [125I]-iodoarylazidoprazosin (IAAP) into ABCB1 in a concentration-dependent manner. In addition, ERK5-IN-1 treatment neither altered the expression level of ABCB1 nor blocked the phosphorylation of downstream Akt or Erk1/2. No significant reversal effect was observed on ABCG2-, ABCC1-, MRP7- and LRP-mediated drug resistance. CONCLUSIONS: Collectively, these results indicated that ERK5-IN-1 efficiently reversed ABCB1-mediated MDR by competitively inhibiting the ABCB1 drug efflux function. The use of ERK5-IN-1 to restore sensitivity to chemotherapy or to prevent resistance could be a potential treatment strategy for cancer patients.

Correction: Alectinib (CH5424802) antagonizes ABCB1- and ABCG2-mediated multidrug resistance in vitro, in vivo and ex vivo.

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