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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. HER2V659E was detected in the plasma of 33% (2/6) of dogs with localized HER2V659E tumors. HER2V659E 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 HER2V659E vs. IC50 > 2,500 nmol/L in HER2WT).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.

HER2 cellular location and function in primary canine lung cancer. (A) Canine papillary adenocarcinoma with intense, complete, circumferential membrane (white arrow) and lateral cytoplasmic membrane (black arrow) anti-HER2 antibody positive staining (brown) in a patient with wild-type HER2. (B) Canine papillary adenocarcinoma with moderate cytoplasmic (black arrow) anti-HER2 antibody positive staining (light brown) in a patient with wild-type HER2. x 40; bar 50 µm. (C) Anti-HER2 immunohistochemistry of a Grade 1 canine papillary adenocarcinoma wild type for HER2. x 20. (D) Segmentation mark-up of the tumor from adjacent normal lung. Tumor is identified by green, whereas red is area within tumor that contains no tissue, and yellow represents areas of non-tumor such as necrosis or tumor stroma. x20.

HER2 expression in primary canine lung cancer based on quantitative real-time PCR analyses. Box plots are shown for HER2 2^(-Î"Î"Ct) expression fold-change relative to the housekeeping gene HPRT (x-axis) in 49 tumors and 14 matched normal lung tissues comparing (A) Normal lung tissue versus tumor tissue, and (B) Normal lung tissue versus HER2-wild-type tumor tissue versus HER2-mutant tumor tissue.

Canine primary lung cancer cell line sensitivity to erlotinib. Five canine cell lines (three HER2WT and two HER2V659E) and one human cell line BT474 (HER2amp) were treated with 10 erlotinib doses ranging from 5x10-8 to 50 μM for 72 hours with CellTiterGlo viability endpoints measured and shown as percent growth inhibition relative to DMSO vehicle control.

Figures S1-S13

Supplementary Data Tables

Canine primary lung cancer cell line sensitivity to lapatinib. Four canine cell lines (three HER2WT and one HER2V659E) were treated with 14 lapatinib doses ranging from 100 μM to 5.5x10-2 nM for 72 hours with CellTiterGlo viability endpoints were measured and shown as percent survival relative to DMSO vehicle control.

Comparison of HER2 mutant allele fractions measured using droplet digital PCR and targeted sequencing in tumor tissue. HER2V659E was evaluated by ddPCR in tumor DNA samples in which these mutations were also detected using amplicon sequencing. We observed a high correlation between allele fractions measured using both techniques (Pearson's r 0.976, p=0.0008).

Supplementary Legend

Extended clinical annotation of primary canine lung cancer patients. Cohort distribution of: (A) Affected breeds, (B) Age at diagnosis, (C) Primary tumor location distribution, (D) Sex, (E) Adenocarcinoma subtype, and (F) Treatment. Yr=years.

Somatic copy number plots derived from exome sequencing of five primary canine pulmonary adenocarcinomas (cPAC) and matched constitutional DNA. Tumor copy number states determined by tCoNutT analysis of tumors and matched constitutional DNA from five cPAC cases is shown with each canine chromosome plotted on the x-axis (shown in alternating green and black) and log2 fold change shown on the y-axis.

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.

Canine primary lung cancer cell line sensitivity to trastuzumab. Five canine cell lines (three HER2WT and two HER2V659E) and one human cell line BT474 (HER2amp) were treated with 10 trastuzumab doses ranging from 1x10-6 μg/ml to 1000 μg/ml for 72 hours with CellTiterGlo viability endpoints measured and shown as percent growth inhibition relative to PBS vehicle control. Relative survival was measured with respect of the sensitive cell line BT474.

Somatic mutation signatures identified by exome sequencing in primary canine lung cancers. (A) The distribution of somatic single nucleotide mutation types in their trinucleotide context from tumor/normal exome sequencing of five cPAC cases. (B) The most common mutation signatures based on trinucleotide context and frequency of somatic single nucleotide mutations from tumor/normal exome sequencing of five cPAC cases. COSMIC Signature 1A (C>T substitutions at NpCpG trinucleotides that are associated with age) was present in four cases. COSMIC Signature 2 (C>T and C>G substitutions at TpCpN, associated with APOBEC cytidine deaminase activity) was present in two cases.

Supplementary Table 1. Informatic tools utilized in primary canine lung cancer analysis Supplementary Table 2. Extended clinical and multiplatform annotation of primary canine lung cancer cases. Supplementary Table 3 : Sequencing metrics for primary canine lung cancer exome analysis. Supplementary Table 4. Somatic coding SNVs identified by exome sequencing of primary canine lung cancers. Supplementary Table 5. Somatic copy number variants identified by exome sequencing in primary canine lung cancers. Supplementary Table 6. Somatic coding structural variants identified by exome sequencing of primary canine lung cancers. Supplementary Table 7. Canine genomic regions covered by custom amplicon panel. Supplementary Table 8. Sequencing metrics for primary canine lung cancer amplicon analysis. Supplementary Table 9. Somatic coding SNVs identified by panel sequencing of primary canine lung cancers. Supplementary Table 10. Germline SNPs in COSMIC Tier 1 cancer genes identified by exome and panel sequencing in primary canine lung cancers. Supplementary Table 11. Multi-platform validation of HER2 mutations. Supplementary Table 12. Non-invasive detection of HER2V659E in the plasma of primary canine lung cancer patients. Supplementary Table 13. HER2 protein expression and quantification by immunohistochemistry.

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.

Background Notch intercellular communication instructs tissue-specific T-cell development and function. In this study, we explored the roles of dendritic cell (DC)-expressed Notch ligands in the regulation of T-cell effector function. Methods We generated mice with CD11c lineage-specific deletion of Notch Delta-like ligand (Dll)1 and Jagged (Jag)2. Using these genetically-ablated mice and engineered pharmacological Notch ligand constructs, the roles of various Delta-like and Jagged ligands in the regulation of T-cell-mediated immunity were investigated. We assessed tumor growth, mouse survival, cytokine production, immunophenotyping of myeloid and lymphoid populations infiltrating the tumors, expression of checkpoint molecules and T-cell function in the experimental settings of murine lung and pancreatic tumors and cardiac allograft rejection. Correlative studies were also performed for the expression of NOTCH ligands, NOTCH receptors and PD-1 on various subsets of myeloid and lymphoid cells in tumor-infiltrating immune cells analyzed from primary human lung cancers. Results Mice with CD11c lineage-specific deletion of Notch ligand gene Dll1, but not Jag2, exhibited accelerated growth of lung and pancreatic tumors concomitant with decreased antigen-specific CD8+T-cell functions and effector-memory (Tem) differentiation. Increased IL-4 but decreased IFN-γ production and elevated populations of T-regulatory and myeloid-derived suppressor cells were observed in Dll1-ablated mice. Multivalent clustered DLL1-triggered Notch signaling overcame DC Dll1 deficiency and improved anti-tumor T-cell responses, whereas the pharmacological interference by monomeric soluble DLL1 construct suppressed the rejection of mouse tumors and cardiac allograft. Moreover, monomeric soluble JAG1 treatment reduced T-regulatory cells and improved anti-tumor immune responses by decreasing the expression of PD-1 on CD8+Tem cells. A significant correlation was observed between DC-expressed Jagged and Delta-like ligands with Tem-expressed PD-1 and Notch receptors, respectively, in human lung tumor-infiltrates. Conclusion Our data show the importance of specific expression of Notch ligands on DCs in the regulation of T-cell effector function. Thus, strategies incorporating selectively engineered Notch ligands could provide a novel approach of therapeutics for modulating immunity in various immunosuppressive conditions including cancer. Electronic supplementary material The online version of this article (10.1186/s40425-019-0566-4) contains supplementary material, which is available to authorized users.

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 non-mutational mechanisms. In this study, we applied this genetic signature to 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.

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. HER2V659E was detected in the plasma of 33% (2/6) of dogs with localized HER2V659E tumors. HER2V659E 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 HER2V659E vs. IC50 > 2,500 nmol/L in HER2WT). 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.

Naturally occurring primary canine lung cancers are aggressive malignancies that are increasingly common in pet dogs. They share clinicopathologic features with human lung cancers in never-smokers, but their genetic underpinnings are unknown. Through multi-platform sequencing of 88 primary canine lung tumors or cell lines, we discovered somatic, coding HER2 (ERRB2) point mutations in 38% of canine pulmonary adenocarcinomas (cPAC, 28/74), but none in adenosquamous (cPASC, 0/11) or squamous cell (cPSCC, 0/3) carcinomas. In cPASC, PTEN was the most frequently mutated gene (18%) while one case each bore likely pathogenic HRAS, KRAS, EGFR, MET, TP53, or VHL somatic mutations. In cPSCC, no recurrently mutated genes were identified, but individual somatic coding mutations were found in BRAF and PTPN11. In cPAC, we also identified recurrent somatic mutation of TP53 (13.5%), SMAD4 (5.4%), PTEN (4.1%), and VHL (2.7%). cPACs assessed by exome sequencing displayed a low somatic mutation burden (median 64 point mutations, 19 focal copy number variants, and 1 translocation). 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 identified in this study were located in the extracellular domain and TMD. HER2V659E was detected in the plasma of 33% (2/6) of dogs with localized HER2V659E tumors. HER2V659E correlated with constitutive phosphorylation of AKT in cPAC cell lines and HER2V659E lines displayed hypersensitivity to the HER2 inhibitors lapatinib and neratinib relative to HER2-wild-type cell lines. These findings have translational and comparative relevance for lung cancer and HER2 inhibition.

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.

LKB1 is functionally lost in 30% of lung adenocarcinoma cases. There are no targeted therapies available for LKB1-loss tumors. Previous studies of LKB1-loss in a mouse model of pancreatic cancer suggested that LKB1 loss drives DNA hypermethylation and serine biogenesis. DNA methyltransferases (DNMTs) maintain CpG methylation, which suppresses gene transcription. The primary substrate for DNMTs is S-adenosyl Methionine (SAMe), and the maintenance of CpG methylation has been shown to be dependent on continued regeneration of SAMe. KRAS status is associated both with LKB1 loss and with a CpG island hypermethylation phenotype (CIMP). Using a previously published 16-gene signature for LKB1 loss, we set out to confirm whether the changes that occur in the mouse model of pancreatic cancer also occur in lung cancer. We applied our signature to samples in The Cancer Genome Atlas (TCGA) lung adenocarcinoma dataset (signature-positive samples are described as “LKB1-loss”), which allowed us to compare both gene expression data as well as methylation data from Illumina 450k microarrays. We then developed a cell-line model for LKB1-loss in order to study the mechanism by which LKB1 regulates DNA methylation. We observed that LKB1-loss is associated with widespread demethylation of the genome in TCGA samples. Approximately 14,000 CpG sites were significantly hypomethylated in LKB1-loss tumors, while only 200 were hypermethylated (adjusted p-value cutoff = 0.05). Furthermore, Student’s t-test showed that LKB1-loss samples have significantly lower average methylation (β-values), regardless of KRAS status. Using motif analysis, we found that demethylated CpG sites are significantly enriched for FOXA1/2/3 consensus binding sites. FOXA transcription factors are well known as pioneering transcription factors that mediate demethylation. We discovered in vitro that FOXA1 localization and turnover is dependent on LKB1 expression, specifically the activity of downstream kinase SIK. Rather than showing enhanced serine biogenesis, LKB1-loss tumors express less DNMT1 and LC-MS/MS analysis shows depletion of SAMe. Finally, in vitro models of LKB1-loss show an increased sensitivity to DNMT inhibitors and FOXA1 silencing. Together, these observations suggest that LKB1-loss lung adenocarcinoma patients have a distinct hypomethylation profile. This profile may make them uniquely sensitive to DNMT-inhibition in combination with other therapies, a hypothesis which we will test in future studies. Citation Format: Michael J. Koenig, Joseph Amann, Bernice Agana, Jacob Kaufman, Vicki Wysocki, Christopher Oakes, David P. Carbone. LKB1 loss in lung adenocarcinoma leads to global hypomethylation and altered FOXA binding [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 824.

Spontaneous primary canine lung cancers are aggressive malignancies that are increasingly common in dogs. They share clinicopathologic features with human lung cancers in never-smokers, but their genetic underpinnings are unknown. As never-smoker lung cancer incidence increases in humans, a need exists for improved biologic understanding and development of new models to fuel translational research. Here, we describe for the first time the detailed clinical and genetic features of primary canine lung cancers through case review and multi-platform sequencing of 90 primary canine lung tumors and cell lines. We performed multi-platform genomic profiling including whole exome sequencing (WES), amplicon panel-based sequencing of 250 regions of 51 cancer genes, and single nucleotide polymorphism (SNP) array-based analysis of tumors and matched constitutional DNA. We profiled 76 cPAC, 11 canine pulmonary adenosquamous carcinomas (cPASC) and three canine pulmonary squamous cell carcinomas (cPSCC). The median age at the time of diagnosis was 11 year old. The most common affected pure breed was the Labrador retriever (19%). Five cPACs assessed by WES displayed low mutation burden with a median of 64 somatic single nucleotide variants (SNVs), 19 somatic copy number variants (CNVs), and 1 structural variant (SV), and frequent C>T substitutions (80%) at NpCpG trinucleotides, a common mutation signature seen across human and canine cancers. Based on WES and amplicon sequencing, we discovered somatic, coding HER2 (ERRB2) point mutations in 37% of cPAC, but none in cPASC or cPSCC. Additional recurrently mutated genes in cPAC included CDKN2A/B (~40%), TP53 (~12%), K/HRAS (~7%), SMAD4 (~5%), and PTEN (~5%). In cPASC, KRAS and TP53 were the most frequently mutated genes (18% each). In cPSCC, no recurrently mutated genes were identified, but individual somatic coding mutations were found in BRAF and PTPN11. The majority (93%) of HER2 mutations were hotspot V659E, located on the transmembrane domain (TMD), and comparable to activating mutations at this same site in human cancer. We were able to detect this mutation not only in biopsied tumors, but also in the plasma of 33% (2/6) of dogs with localized V659E-positive tumors. Other somatic HER2 mutations identified were similarly located in the HER2 juxtamembrane domain and TMD including A664T and K676E. HER2 point mutations occurred in the absence of significant focal amplification (assessed by WES and SNP array) or overexpression (assessed by qRT-PCR and IHC). We also showed that HER2 mutation correlated with constitutive phosphorylation of Akt in cPAC cell lines. Furthermore, HER2 V659E-mutant lines displayed hypersensitivity to the HER2 kinase inhibitors neratinib (IC50, 23nM) and lapatinib (IC50, 168nM) relative to HER2 wild-type cell lines (IC50, >2500nM). These data bear implications for comparative understanding of HER2-mutant lung cancer across species. Citation Format: Gwendolen Lorch, Karthigayini Sivaprakasam, Victoria Zissman, Nieves Perdigones, Tania Contente-Cuomo, Alexandra Nazareno, Salvatore Facista, ShukMei Wong, Winnie Liang, Joseph M. Amann, Sara L. Sinicropi-Yao, Michael J. Koenig, Krista La Perle, Timothy G. Whitsett, Muhammed Murtaza, Jeffrey Trent, David P. Carbone, William P. Hendricks. Identification of frequent HER2 activating mutations in canine primary pulmonary adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3705.

Lung cancer is the leading cause of cancer-related death in the United States and worldwide. Novel therapeutic developments are critically necessary to improve outcomes for this disease. Aberrant epigenetic change plays an important role in lung cancer development and progression. Therefore, drugs targeting the epigenome are being investigated in the treatment of lung cancer. Monotherapy of epigenetic therapeutics such as DNA methyltransferase inhibitors (DNMTi) and histone deacetylase inhibitors (HDACi) have so far not shown any apparent benefit while one of the clinical trials with the combinations of DNMTi and HDACi showed a small positive signal for treating lung cancer. Combinations of DNMTi and HDACi with chemotherapies have some efficacy but are often limited by increased toxicities. Preclinical data and clinical trial results suggest that combining epigenetic therapeutics with targeted therapies might potentially improve outcomes in lung cancer patients. Furthermore, several clinical studies suggest that the HDACi vorinostat could be used as a radiosensitizer in lung cancer patients receiving radiation therapy. Immune checkpoint blockade therapies are revolutionizing lung cancer management. However, only a minority of lung cancer patients experience long-lasting benefits from immunotherapy. The role of epigenetic reprogramming in boosting the effects of immunotherapy is an area of active investigation. Preclinical studies and early clinical trial results support this approach which may improve lung cancer treatment, with potentially prolonged survival and tolerable toxicity. In this review, we discuss the current status of epigenetic therapeutics and their combination with other antineoplastic therapies, including novel immunotherapies, in lung cancer management.

We studied the ranking of osteoporosis (OP) medication attributes in a convenience sample of four different racial/ethnic groups in the United States. Our study showed that postmenopausal women differ in the ranking of OP medication attributes based on age, educational level, income, and prior fracture history. Decision making about OP medication-related behavior relies heavily on patient preferences about specific medication attributes. Patients may decide to initiate, change, or stop therapies based on ranking of perceived attributes of the therapy and their personal attitudes toward those attributes. We used MaxDiff, a form of conjoint analysis (Ryan and Farrar 2000), to explore patient weighting of attributes across four racial/ethnic groups at two sites in the United States and defined four critical attributes that influence such decisions (safety, efficacy, cost, and convenience) from qualitative interviews. We recruited a sample of 367 Postmenopausal (PM) women at risk of OP fractures from four racial/ethnic groups: Caucasian (n = 100), African American (n = 100), Asian American (n = 82), and Hispanic American (n = 85). Respondents completed a laptop-based questionnaire that included demographic items, several short scales on medical care preference and OP patient perceptions, and a MaxDiff procedure that determines comparative ranking of attributes either as least important or most important to their decisions. MaxDiff analyses were done to evaluate the relative weight of specific statements for each participant and to determine whether racial/ethnic groups differed across dimensions. Overall, participants in all four groups rated efficacy > safety > cost > convenience. Although there were no significant differences among the racial/ethnic groups on overall ranking of attributes, subgroup analyses revealed significant impact of age, education, income, and prior fracture on these decisions. The findings from this study suggest that postmenopausal women differ in their ranking of OP medication attributes, and healthcare providers must account for personal preferences in their communication about and selection of OP medications.

The serine-threonine liver kinase B1 (LKB1, also called STK11) is a tumor suppressor that functions as master regulator of cell growth, metabolism, survival and polarity. Germline mutation of LKB1 causes Peutz-Jeghers syndrome, which is an autosomal dominant disease characterized by mucocutaneous pigmentation and hamartomatous polyps. In non-small-cell lung cancer (NSCLC), LKB1 is frequently rendered non-functional, either through mutation or down regulation. As a tumor suppressor and a protein whose function is lost, identifying pathways that are activated with LKB1 loss may be the only way to target such tumors. Some previous studies have suggested that inhibition of mTOR, a growth promoting pathway negatively regulated by LKB1, as a potential target in LKB1 mutant NSCLC. In this study, we investigated the effect of LKB1 activity on the sensitivity to a PI3K/mTOR inhibitor using four LKB1 mutant NSCLC cell lines to which LKB1 gene was transduced and one LKB1 wild type NSCLC cell line in which LKB1 gene was knocked out by CRISPR technology. Transduction of LKB1 resulted in significant resistance to a PI3K/mTOR inhibitor in two of the four LKB1 add-back cell lines, while knocking out LKB1 in the LKB1 wild type cell line induced PI3K/mTOR inhibitor sensitivity. The mechanism behind these observed results appears to be through regulation of phosphorylation of AKT. The presence of LKB1 led to a persistent AKT phosphorylation even in the presence of the PI3K/mTOR inhibitor, both in add back cell and wild type cells. Our data suggest that the identification of LKB1 activity may be a promising biomarker for the sensitivity to PI3K/mTOR inhibition. Further investigation into the mechanism of LKB1 loss and increased sensitivity to inhibition of the PI3K and mTOR pathways is warranted. Citation Format: Takehito Shukuya, Tadaaki Yamada, Michael J. Koenig, Mohammad A. Rahman, Joseph M. Amann, David P. Carbone. Effect of LKB1 activity on the sensitivity to PI3K/mTOR inhibitor in non-small-cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5535. doi:10.1158/1538-7445.AM2017-5535

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.

Chymotrypsin activity is rapidly inactivated by the N-mustard anti-tumor drug, chlorambucil. Since mustards react with thiols, amines, carboxyls, imidazoles, and sulfide sites on proteins, N-acetylcysteine, 2 proprietary protein hydrolyzates, beta-mercaptoethanol, ethanolamine, and sodium lactate were tested for their capacity to protect chymotrypsin from inactivation by the mustard. In each instance, protection was afforded to chymotrypsin. In as much as N-acetylcysteine protected chymotrypsin from inactivation by chlorambucil, it is suggested that this thiol compound may serve as a detoxication agent and may not require prior transformation into glutathione by cells in order to reduce mustard levels within the cells, as suggested by Smith and Gross (Proceedings of the NATO Panel VIII meeting, Grenoble, France, 1991.) It is further suggested that amino acids present as biosynthetic and degradative components of cells may detoxify mustards.