STK11/LKB1 Loss of Function Is Associated with Global DNA Hypomethylation and S-Adenosyl-Methionine Depletion in Human Lung Adenocarcinoma.

STK11 (liver kinase B1, LKB1) is the fourth most frequently mutated gene in lung adenocarcinoma, with loss of function observed in up to 30% of all cases. Our previous work identified a 16-gene signature for LKB1 loss of function through mutational and nonmutational mechanisms. In this study, we applied this genetic signature to The Cancer Genome Atlas (TCGA) lung adenocarcinoma samples and discovered a novel association between LKB1 loss and widespread DNA demethylation. LKB1-deficient tumors showed depletion of S-adenosyl-methionine (SAM-e), which is the primary substrate for DNMT1 activity. Lower methylation following LKB1 loss involved repetitive elements (RE) and altered RE transcription, as well as decreased sensitivity to azacytidine. Demethylated CpGs were enriched for FOXA family consensus binding sites, and nuclear expression, localization, and turnover of FOXA was dependent upon LKB1. Overall, these findings demonstrate that a large number of lung adenocarcinomas exhibit global hypomethylation driven by LKB1 loss, which has implications for both epigenetic therapy and immunotherapy in these cancers. SIGNIFICANCE: Lung adenocarcinomas with LKB1 loss demonstrate global genomic hypomethylation associated with depletion of SAM-e, reduced expression of DNMT1, and increased transcription of repetitive elements.

Identification of Recurrent Activating HER2 Mutations in Primary Canine Pulmonary Adenocarcinoma.

PURPOSE: Naturally occurring primary canine lung cancers share clinicopathologic features with human lung cancers in never-smokers, but the genetic underpinnings of canine lung cancer are unknown. We have charted the genomic landscape of canine lung cancer and performed functional characterization of novel, recurrent HER2 (ERBB2) mutations occurring in canine pulmonary adenocarcinoma (cPAC). EXPERIMENTAL DESIGN: We performed multiplatform genomic sequencing of 88 primary canine lung tumors or cell lines. Additionally, in cPAC cell lines, we performed functional characterization of HER2 signaling and evaluated mutation-dependent HER2 inhibitor drug dose-response. RESULTS: We discovered somatic, coding HER2 point mutations in 38% of cPACs (28/74), but none in adenosquamous (cPASC, 0/11) or squamous cell (cPSCC, 0/3) carcinomas. The majority (93%) of HER2 mutations were hotspot V659E transmembrane domain (TMD) mutations comparable to activating mutations at this same site in human cancer. Other HER2 mutations were located in the extracellular domain and TMD. HER2 V659E was detected in the plasma of 33% (2/6) of dogs with localized HER2 V659E tumors. HER2 V659E cPAC cell lines displayed constitutive phosphorylation of AKT and significantly higher sensitivity to the HER2 inhibitors lapatinib and neratinib relative to HER2-wild-type cell lines (IC50 < 200 nmol/L in HER2 V659E vs. IC50 > 2,500 nmol/L in HER2 WT). CONCLUSIONS: This study creates a foundation for molecular understanding of and drug development for canine lung cancer. These data also establish molecular contexts for comparative studies in dogs and humans of low mutation burden, never-smoker lung cancer, and mutant HER2 function and inhibition.

The Effect of LKB1 Activity on the Sensitivity to PI3K/mTOR Inhibition in Non-Small Cell Lung Cancer.

INTRODUCTION: Liver kinase B1 (LKB1), also called serine/threonine kinase 11 (STK11), is a tumor suppressor that functions as master regulator of cell growth, metabolism, survival, and polarity. Approximately 30% to 35% of patients with NSCLC possess inactivated liver kinase B1 gene (LKB1), and these patients respond poorly to anti-programmed cell death 1 (PD-1)/programmed death ligand 1 (PD-L1) immunotherapy. Therefore, novel therapies targeting NSCLC with LKB1 loss are needed. METHODS: We used a new in silico signaling analysis method to identify the potential therapeutic targets and reposition drugs by integrating gene expression data with the Kyoto Encyclopedia of Genes and Genomes signaling pathways. LKB1 wild-type and LKB1-deficient NSCLC cell lines, including knockout clones generated by clustered regularly interspaced short pallindromic repeats-Cas9, were treated with inhibitors of mechanistic target of rapamycin kinase (mTOR) and phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) and a dual inhibitor. RESULTS: In silico experiments showed that inhibition of both mTOR and PI3K can be synergistically effective in LKB1-deficient NSCLC. In vitro and in vivo experiments showed the synergistic effect of mTOR inhibition and PI3K inhibition in LKB1-mutant NSCLC cell lines. The sensitivity to dual inhibition of mTOR and PI3K is higher in LKB1-mutant cell lines than in wild-type cell lines. A higher compensatory increase in Akt phosphorylation after rapamycin treatment of LKB1-deficient cells than after rapamycin treatment of LKB1 wild-type cells is responsible for the synergistic effect of mTOR and PI3K inhibition. Dual inhibition of mTOR and PI3K resulted in a greater decrease in protein expression of cell cycle-regulating proteins in LKB1 knockout cells than in LKB1 wild-type cells. CONCLUSION: Dual molecular targeted therapy for mTOR and PI3K may be a promising therapeutic strategy in the specific population of patients with lung cancer with LKB1 loss.

Rapid and Highly Sensitive Detection of Therapeutically Relevant Oncogenic Driver Mutations in EBUS-TBNA Specimens From Patients With Lung Adenocarcinoma.

BACKGROUND: First-line afatinib treatment prolongs overall survival in patients with metastatic non-small-cell lung cancer (NSCLC) harboring exon 19 deletion of epidermal growth factor receptor (EGFRdelEx19) mutations. In contrast, Kirsten rat sarcoma 2 viral oncogene homolog (KRAS) mutations are negative predictors for benefit from EGFR-targeting agents. Endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) is well-established for lung cancer diagnosis and staging. Next generation sequencing (NGS) allows for simultaneous interrogation for multiple mutations but has limitations (required tumor tissue amount, assay times). Reverse transcription polymerase chain reaction (RT-PCR) using light-Cycler technology (LCRT-PCR) can rapidly and sensitively detect somatic mutations from NSCLC patients. In the present study, we analyzed the feasibility of LCRT-PCR for rapid EGFRdelEx19 and KRAS exon 2 mutation detection in EBUS-TBNA samples and compared the LCRT-PCR and NGS results. MATERIALS AND METHODS: A total of 48 EBUS-TBNA samples from 47 patients with a confirmed diagnosis of pulmonary adenocarcinoma were analyzed using LCRT-PCR (as previously described) and NGS (MiSeq; Illumina) using targeted resequencing and a customized multiplex PCR panel. The processing time was ∼1 week for the NGS and < 24 hours for the LCRT-PCR analyses. RESULTS: All (100%) EGFRdelEx19 and KRAS exon 2 mutations detected by NGS were detected by LCRT-PCR. In addition, LCRT-PCR detected 2 KRAS exon 2 mutations and 3 EGFRdelEx19 mutations that were not detected by NGS. CONCLUSION: LCRT-PCR is a highly sensitive method to rapidly detect mutations of therapeutic relevance (eg, EGFRdelEx19 and KRAS exon 2) in EBUS-TBNAs from NSCLC patients. It is of value as an initial assay for first-line treatment decisions.

Assessment of the risk of needlestick injuries associated with intravitreal injections.

PURPOSE: To assess the overall risk of needlestick injuries (NSIs) associated with intravitreal injection, and more specifically related to the practice of compounding pharmacies of applying informational adhesive stickers to repackaged bevacizumab injection syringes. METHODS: This cross-sectional study involved an online survey of retina specialists in the United States. RESULTS: Of the 717 invited retina specialists, 158 (22%) responded to the online survey. The respondents reported using 1 pair of gloves (51%), no gloves (46%), or 2 pairs of gloves (3%) during intravitreal injection. Repackaged bevacizumab syringes distributed by compounding pharmacy were used by 89% of the respondents, and 63% reported that the adhesive sticker was applied directly to the syringe. Unintentional adhesion between the sticker and hand or glove was experienced by 9% of respondents. At least 1 NSI during intravitreal injection was experienced by 8% of respondents, and sticker-related injury was reported by 3%. The sticker was perceived to increase risk for NSI by 33% of respondents. CONCLUSION: This survey showed that 8% of the responding physicians surveyed have experienced at least one NSI during intravitreal injections, of which approximately one third was attributed to the adhesive sticker. Direct application of misfitting stickers to repackaged syringes by compounding pharmacies may be a practice that can aggravate the risk of NSI.