Citations

Abstract Hepatocellular carcinoma (HCC), or liver cancer, is the second leading cause of death from cancer worldwide (Ferlay et al, Lyon, France: International Agency for Research on Cancer, 2013). HCC is linked with hepatic steatosis, or fatty liver disease, the most common chronic liver disease in the US. Both hepatic steatosis and HCC are increasing in prevalence in the US. However, the molecular mechanisms that cause HCC in a steatotic liver are not entirely understood. To uncover mutations promoting cancer in fatty livers and elucidate the role of steatosis in HCC, we performed a forward genetic screen using the conditional Sleeping Beauty (SB) transposon insertional mutagenesis system in mice treated with ethanol and a choline deficient diet to induce steatosis. Mice developed hepatic steatosis and liver cancer without sex bias, modeling the pattern seen in humans in whom HCC's with steatosis as a preceding condition are represented by a nearly equal number of males and females. We found that hepatic steatosis caused a downregulation of liver Foxa1 expression and increased Foxa2 inactivating phosphorylation, thus providing a molecular explanation for the loss of gender bias in our mouse model. Transposon insertion sites were mapped from the mouse HCC tumors, and genes near or in which more insertions were observed than expected by chance were identified as common insertion sites (CIS), which we consider putative steatosis-associated liver cancer genes. Wnt/b-catenin signaling, which defines a subclass of HCC and can induce tumors in mice without sex bias, was one of the top CIS associated pathways identified. Interestingly, alpha-adrenergic and dopamine signaling were also among the top steatosis-specific CIS associated pathways, suggesting a role for the sympathetic nervous system and cAMP signaling in fatty liver-associated cancer. Further studies are underway to test the roles of the pathways and individual genes identified in this study in driving HCC. Our studies implicate Wnt/beta-catenin and sympathetic nervous system signaling in steatosis-associated HCC and suggest methods for treatment or prevention in such cases. Citation Format: Barbara R. Tschida, N. Alpay Temiz, Ju D. Yang, Vincent W. Keng, David A. Largaespada. A transposon-based forward genetic screen for steatosis-associated liver cancer drivers. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 63.

Abstract BACKGROUND: We previously showed that mRNA expression of squalene monooxygenase (SQLE), part of the cholesterol synthesis pathway, is associated with lethal prostate cancer. In-vitro studies suggest that loss of PTEN expression and resulting PI3K pathway activation drive cholesterol ester accumulation in aggressive prostate cancers through sterol regulatory element-binding protein (SREBP) and acyl coenzyme A-cholesterol acyltransferase (ACAT1) activity. In two prospective cohorts, we studied whether lower PTEN expression is associated with cholesterol metabolism and how this relates to lethal prostate cancer. METHODS: We analyzed men with prostate cancer from the prospective prostatectomy Health Professionals Follow-up Study and Physicians’ Health Study. 105 men had lethal cancer and 284 men non-lethal disease without metastases at 8 years of follow-up. Whole-transcriptome mRNA expression profiling data was available from diagnostic prostate tumor specimens. Linear regression models were used to assess continuous and categorical associations, and Pearson correlation coefficients were calculated. Logistic regression was used to obtain odds ratios (ORs) and 95% confidence intervals (CIs) of lethal prostate cancer. RESULTS: Both lower PTEN and higher SQLE expression were associated with higher Gleason grade (p trend < 0.001 each). Correlations of PTEN with SREBF1 (r = -0.08, p = 0.1), SREBF2 (r = -0.09, p = 0.07), and ACAT1 (r = 0.04, p = 0.4) were weak. SREBF2 was correlated with SQLE (r = 0.33, p < 0.001) but not with ACAT1 (r = -0.01, p = 0.8). Low PTEN was associated with lethal cancer (OR, 1.50 per 1 standard deviation [SD] decrease; 95% CI, 1.20 to 1.87; p = 0.001). High SQLE was associated with lethal cancer (OR 2.13 per 1 SD increase, 95% CI, 1.63 to 2.79; p < 0.001). SREBF1, SREBF2, and ACAT1 were not associated with lethal cancer (all p > 0.05). Adjusting for PTEN mildly attenuated the association of SQLE with lethal cancer (OR, 2.07; 95% CI, 1.57 to 2.73), as did additional adjustment for age, Gleason grade, and stage (SQLE: OR, 1.75; 95% CI, 1.29 to 2.40). In the lowest quartile of PTEN expression, SQLE was less strongly associated with lethal cancer (OR 1.74; 95% CI, 1.20 to 2.52) compared to the upper three quartiles (OR 2.47; 95% CI, 1.69 to 3.60; p interaction = 0.2). CONCLUSION: Low PTEN mRNA expression and high SQLE are features of aggressive prostate cancers. However, at the time of prostatectomy, SREBF1/2 expression and resulting ACAT1 expression do not appear to be major characteristics of prostate cancers with lethal outcomes. Citation Format: Konrad H. Stopsack, Travis A. Gerke, Lorelei A. Mucci, Jennifer R. Rider. PTEN expression, cholesterol metabolism, and lethal prostate cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 60.

Abstract Introduction: Multiple myeloma (MM) is a malignancy of plasma cells that is still considered incurable due to the 90% relapse rate in patients. Despite the advent of new agents the majority of MM patients relapse secondary to therapy resistance. The potential of reovirus (RV) as a novel therapeutic agent for MM under in vitro, in vivo and ex vivo conditions has been demonstrated by us and a phase I clinical trial of MM with RV therapy has shown indications of efficacy. Recently we have shown that RV could synergize with several standard of care MM drugs such as bortezomib (BTZ), carfilzomib (CFZ) and dexamethasone and importantly, enhance its therapeutic potential in therapy resistant human MM cell lines in vitro. Utilizing the syngeneic Vk*MYC BTZ resistant transplantable MM mouse model, we demonstrate that mice harbouring BTZ insensitive MM tumors significantly respond to RV treatment. Our data indicate that this RV treatment sensitivity is manifested via, direct oncolysis in conjunction with immune modulatory effects. Methods: Vk*MYC myeloma cells (Vk12598) were injected via tail vein (IV) into 3 groups of C57BL/6 wt recipient mice. Seven days post tumor injection, mice were treated with PBS (vehicle) or live (LV) or dead reovirus (DV) (5×10⁁8 PFU) administered IV every 6 days in a total of 5 doses. Serum gamma globulins (M-spike) were assessed weekly by serum protein electrophoresis. Mice were sacrificed on day 35 post tumour injection and their spleens and bone marrow (BM) were harvested. Splenic and BM cells were stained for CD45+/CD45R-/CD138+ (MM tumors), CD4+/CD8+ (T cells), CD3+/NKG2D+ (NKT cells), CD11b+Ly6C+ (monocytic myeloid derived suppressor cells (Mo-MDSC) and CD11b+Ly6G+ (polymorphonuclear (PMN) - MDSCs) and analyzed by flow cytometry. Results: Mice treated with RV demonstrated highly significant (P<0.001) reductions in their M-spikes at 4 and 5 weeks post treatment. RV treatment led to a significant NKT cell accumulation in splenic and BM of RV treated mice in comparison to vehicle treated mice suggestive of immune activation due to RV. Further, RV treatment led to a significant increase in BM Mo-MDSC number but not PMN-MDSC numbers. Interestingly, significant down regulation of splenic PMN-MDSCs was seen with RV treatment. Conclusions: Our results indicate reovirotherapy is successful in a validated syngeneic BTZ resistant MM model. These results have important implications for future clinical trials. Currently we are conducting further experiments to assess the immune modulatory (such as T - memory and T - regulatory cells) contributions of RV in this model. Citation Format: Chandini M. Thirukkumaran, Zhong Qiao Shi, Joanne Luider, Karen Kopciuk, Marta Chesi, Leif Bergsagel, Yuan Dong, Chunfen Zhang, Ahmed Mostafa, Kathy Gratton, Satbir Thakur, Don Morris. Successful oncolytic virotherapy in a bortezomib resistant syngeneic mouse model of multiple myeloma: Implications for translational significance. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 541.

Abstract The completion of The Cancer Genome Atlas (TCGA) project for colorectal cancer (CRC) has enabled a new, focused phase of sequencing tumor samples for which genome-wide genetic, epidemiological, clinical and lifestyle data have been collected. By combining somatic mutational profiles with these aforementioned data, we may identify and report effective prevention and treatment approaches for a broader population of individuals. The advent of targeted deep sequencing approaches using DNA obtained from formalin fixed paraffin embedded tissues has made possible the genetic characterization of the large numbers of patients needed to make such an effort relevant. As a first step, we describe a custom gene panel designed from large-scale studies for targeted deep sequencing, and its application to over 4,200 CR tumors, collected by the Genetics and Epidemiology of Colorectal Cancer Consortium (GECCO). This study is designed to identify recurrent somatic mutations and copy number alterations (CNAs) for association testing with germline genetic and lifestyle and environmental risk factors for CRC, and thereby identify relevant approaches to impact cancer prevention. The targeted CRC panel includes 205 genes. Genes were primarily selected as significantly mutated genes identified from the Nurses’ Health Study and Health Professional's Follow-up Study (n = 700), and TCGA (n = 525). We also included 15 genes associated with high penetrance germline mutations and augmented the list to include genes in pathways of somatically altered genes, identified by literature review, from public databases and known to be associated with loss of heterozygosity. For these 205 genes, amplification primers were designed to include all coding regions of transcripts that are listed in the UCSC Genome Browser. For regions with CNAs, the TCGA dataset was analyzed to include regions with greater than or equal to 2 copy focal gains or losses that were found in more than 4 or 3 tumors, respectively. Candidate genes in regions with significant CNAs from the TCGA CRC publication (Nature 2012) were also included. For CNAs, 6 to 12 amplicons were designed for each of the 56 selected regions (32 gains and 24 losses). Additional target regions include: 1) 25 microsatellite loci previously used to identify defective DNA mismatch repair and 212 homoploymer repeats. These include microsatellite loci recommended by the NCI Consensus Panel for identifying MSI; 2) amelogenin (for gender); and 3) Fusobacterium to detect a putative CRC-associated pathogen in tumor biopsies. At the AACR annual meeting, we expect to present results for deep sequencing (∼1,000X) of the first 1,000 CR tumors, including any preliminarily identified pathways and subtypes that may provide the basis for association testing with germline genetic and lifestyle and environmental risk factors needed for inferring better approaches to prevention and treatment of CRC. Citation Format: Syed H. Zaidi, Catie Grasso, Jasmine Mu, Eve Shinbrot, Marios Giannakis, Charles Connolly, Ivan Borozan, Hermann Brenner, Peter Campbell, Andrew Chan, Jenny Chang-Claude, Mengmeng Du, Vincent Ferretti, Amy French, Charles Fuchs, Steven Gallinger, Levi Garraway, Andrea Gsur, Marc Gunter, Tabitha Harrison, Michael Hoffmeister, Li Hsu, Wen-Yi Huang, Jeroen Huyghe, Mathieu Lemire, Elaine Mardis, John McPherson, Polly Newcomb, Lincoln Stein, Wei Sun, Lee Timms, Quang Trinh, David Wheeler, Christina Yung, Niha Zubair, Shuji Ogino, Stephen Thibodeau, Ulrike Peters, Thomas Hudson. Linking the molecular profile of colorectal tumors to germline genetic and environmental risk factors. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5221.

Abstract Sequencing tests assaying panels of genes or whole exomes are widely available for cancer risk evaluation. However, methods for classification of variants resulting from this testing are not well studied. We evaluated the ability of American College of Medical Genetics and Genomics (ACMG) guidelines to define the rate of mutations and variants of uncertain significance (VUS) in 180 medically relevant genes, including all ACMG designated reportable cancer and non-cancer genes, in individuals who met guidelines for hereditary cancer risk evaluation. We performed whole exome sequencing in 404 individuals in 257 families and classified 1640 variants from these genes. Potentially clinically actionable (likely-pathogenic/pathogenic, LP/P) versus nonactionable (VUS/likely-benign/benign) calls were 92% and 88% concordant with locus specific databases and Clinvar, respectively. LP/P mutations were identified in 11 of 25 breast cancer susceptibility genes in 27 BRCA1/2 negative families (11%). Evaluation of 84 additional autosomal dominant cancer susceptibility genes identified LP/P mutations in only four additional families (1.7%), suggesting they do not influence risk in this cohort. However, individuals from nine of 257 families (3.5%) had incidental LP/P mutations in 32 non-cancer disease genes, and 7% of individuals were monoallelic carriers of an LP/P mutation in 39 autosomal recessive cancer syndrome genes. Furthermore, 90% of individuals had at least one VUS. In summary, these data support the clinical utility of ACMG variant classification guidelines. In addition, evaluation of extended panels of cancer genes in breast/ovarian cancer families leads to only an incremental clinical benefit but substantially increases the complexity of the results. Citation Format: Kara N. Maxwell, Steven N. Hart, Joseph Vijai, Kasmintan A. Schrader, Tinu Thomas, Bradley Wubbenhorst, Vignesh Ravichandran, Raymond M. Moore, Chunling Hu, Lucia Guidugli, Brandon Wenz, Thomas P. Slavin, Susan M. Domchek, Mark E. Robson, Csilla Szabo, Susan L. Neuhausen, Jeffrey N. Weitzel, Kenneth Offit, Fergus J. Couch, Katherine L. Nathanson. Evaluation of ACMG guideline classified variants in 180 cancer and incidental non-cancer genes in families with breast/ovarian cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5220.

Abstract This study investigates how increased stiffness of the tumor microenvironment (TME) can facilitate cellular multinucleation. Multinucleation is an easily observable marker for polyploidy, facilitating the quantification of this phenotype. Polyploidy is commonly an intermediate to aneuploidy, defined as an abnormal number of chromosomes, a phenotype found in 85% of solid cancers that can drive tumorigenesis. Up to 37% percent of tumors exhibit whole-genome doubling, which typically precedes other somatic copy number alterations. Additionally, induction of tetraploidy in human cells promoted increased tolerance for mutation, resistance to chemotherapeutic drugs, and transformation in culture. Tumors are inherently stiffer than normal tissue, and this property has been shown to affect cell growth and proliferation. Similarly, cell cycle errors have long been linked to chromosomal abnormalities. Here, we used engineered two-dimensional substrata that mimic tumor and normal microenvironments to investigate how matrix stiffness regulates multinucleation in mammary epithelial cells. Cells cultured on “stiff” substrata, representing tumor tissue, showed a nearly 14-fold increase in multinucleation compared to cells cultured on “soft” substrata, representing normal tissue. We found that multinucleation was regulated in part by signaling downstream of MMP3, a protease commonly upregulated in cancer. This signaling depended on expression of the Rac1 splice variant, Rac1b, production of reactive oxygen species (ROS), and expression of Snail. Under all conditions, cells cultured on soft substrata maintained a low frequency of multinucleation. We also investigated biochemical effectors downstream of Snail. Aurora A kinase (Aurora A) is a mitotic regulator commonly elevated in cancer that has been shown to induce multinucleation in mammary epithelial cells and increase tumor incidence in mice. We found using microarray analysis that MMP3 increases the levels of Aurora A in mammary epithelial cells. We further discovered that upregulation of Aurora A in response to MMP3 signaling was limited to one transcript variant. These data suggest that an increase in multinucleation is driven by the stiffening of the tissue during tumorigenesis, in part by MMP3-induced signaling and regulation of Aurora A splicing. More broadly, these data suggest a key role for the mechanical properties of the tumor microenvironment in cancer progression. Citation Format: Allison K. Simi, Derek C. Radisky, Celeste M. Nelson. Tissue stiffness regulates multinucleation in mammary epithelial cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5097.

Abstract Background: KRAS gene mutations are present in approximately 40% of colorectal adenocarcinomas and predict for nonresponse to the anti-epidermal growth factor receptor antibodies used in routine clinical practice. The most common activating mutations in this malignancy occur in codons 12 and 13 of exon 2. Determination of KRAS mutational status from tumor samples is an essential tool for managing patients with colorectal cancer (CRC) but requires tissue obtained by invasive percutaneous or surgical biopsies. Recent advances allow for the isolation and analysis of cell-free DNA (cfDNA) from the peripheral blood. A highly sensitive multiplex technology that uses low amounts of input DNA is needed to establish cfDNA as a liquid biopsy to personalize care. Methods: Digital droplet PCR (ddPCR) is a particularly sensitive method for detecting rare and low copy targets. Related work led to 7 individual assays, and to companion 4-plex and 3-plex assays, to accommodate the 7 common KRAS mutations and internal controls. A single 7-plex assay is favored to maximize efficiency and minimize cost. We optimized the RainDrop Digital PCR System to collectively detect the G12/G13 KRAS mutations, given that documentation of a mutation (not the specific mutation) is the factor of clinical relevance. Commercially available cell lines and genomic DNA aliquots were used as reference standards and for determinations of sensitivity and limit of blank (LOB). Initial clinical validation samples included tumor DNA derived from FFPE tissue (n = 10), cfDNA from healthy donors (n = 10), and cfDNA from metastatic CRC patients with tumors of known KRAS mutation status (n = 4). All assays were run in triplicate to confirm reproducibility. Results: Custom Taqman MGB probes were selected for KRAS wild type (WT), G12A, G12C, G12D, G12R, G12S, G12V, and G13D to allow for testing in a single reaction. LOB was 9 per 1×10(6) droplets on the basis of WT controls, including cfDNA samples from healthy donors. Evaluation of cell line DNA, genomic DNA aliquots, and FFPE samples demonstrates that the assay detects mutant KRAS with 100% accuracy and 0.1% mutant DNA in a total input of 10 ng WT/mutant DNA. With the clinical samples, concordant KRAS findings between cfDNA and tissue were obtained for two patients with WT tumors and one patient with a KRAS G12S mutant tumor. cfDNA analysis did not detect the G12V mutation identified in the colon primary of the other patient. Evaluation of additional clinical samples is in progress. Conclusions: Our 7-plex G12/G13 KRAS assay has excellent accuracy and is able to detect the KRAS mutations of interest at low (0.1%) mutation frequencies. Additional studies are being performed on prospectively collected plasma samples from patients with metastatic CRC of known KRAS mutation status. Large scale correlation between tumor mutation status and cfDNA findings will be performed to validate our assay for clinical implementation. Citation Format: Rajeswari Avula, Benjamin R. Kipp, Jesse S. Voss, Keegan E. Haselkorn, W. Edward Highsmith, Kevin C. Halling, Jeremy C. Jones, Steven R. Alberts, Michael B. Campion, Cassandra J. Nelson, Jin Jen, Eric D. Wieben, Julie M. Cunningham, Minetta C. Liu. Optimized 7-Plex cell-free DNA assay for clinically relevant KRAS mutations in colorectal cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 505.

Abstract Introduction: The RAS signaling pathway is an important growth-promoting pathway that controls cell proliferation and survival in multicellular organisms. During oncogenesis, activation of EGFR/HER2/K-RAS signaling pathway reduces focal adhesion, increases cell motility, promote tumorigenesis and accelerate tumor invasion and metastasis. SIAH2, which is the human homologue of a highly conserved family of RING-domain E3 ubiquitin ligase, is a key downstream signaling component which is essential for RAS signal transduction. We noticed that SIAH2 can regulate cell migration, tumorigenesis and metastasis by unknown mechanisms. Methods: Using HIV-based lentiviral system, we introduced an anti-SIAH molecule, the proteolysis-deficient mutant form of SIAH2 (SIAH2PD), into multiple types of human cancer cell lines. The changes of cell morphology, focal adhesion and adherens junction were examined by immunofluorescence (IF) staining. Images were captured on day 3 post infection experiments to show the changes in cell focal adhesion/junction. Using lentiviral system, we introduced exogenous LIM-domain proteins into human cancer cell lines which already expressed SIAH2PD, and then check the cell focal adhesion rescue by immunofluorescence staining. Results: We identified and validated three proteins that interact with SIAH2, contain LIM domains, and regulate focal adhesion and cell attachment. Through in vitro and in vivo experiments, we show that SIAH2 binds, ubiquitinates and degrades thyroid receptor-interacting protein 6 (TRIP6), Four-and-a-half LIM domain protein 2 (FHL2) and Leupaxin (LPXN), indicating that these evolutionarily conserved LIM-domain proteins are bona fide SIAH2 substrates and their double zinc finger motif might serve as a degron signal for SIAH2 E3 ligase. Our staining results showed that SIAH-deficiency would disrupt Trip6/FHL2/LPXN localization at the focal adhesion site, and inhibit cell motility. By introducing exogenous Trip6/FHL2/LPXN proteins into cancer cells, the focal adhesion deficiency and cell death caused by SIAHPD can be rescued, and the ability of cell migration and tumor initiation can be restored. Conclusions: We show that in cancer cells, SIAH2 antagonizes TRIP6, FHL2 and LPXN in modulating focal adhesions and cell motility. This interaction could be important in cell dissemination, invasion and metastasis. The suppression of cell motility and tumorigenesis in response to SIAH-deficiency could provide a new molecular mechanism to reveal the importance of RAS/SIAH signaling pathway in cancer cells. By circumventing this essential cell growth signaling pathway in cancer, we hope to develop novel anti-SIAH-based therapeutic drugs, which may help to inhibit cancer cell dissemination, invasion and metastasis. Citation Format: Minglei Bian, Yang Liao, Monicah Njogu, Rebecca Schmidt, Rie Takahashi, Zandra Walton, Amy H. Tang. SIAH2 E3 ligase targets LIM-domain proteins for degradation to modulate focal adhesion and cell motility. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5049.

Abstract Background: We recently have reported that co-mutations within KRAS-mutated lung adenocarcinoma (LUAD) define a distinct biological phenotype and may offer insights into therapeutic targeting using this classification system. The analysis revealed striking differences in checkpoint ligand expression and T cell infiltration between TP53 and STK11 (LKB1) co-mutated KRAS LUAD. We, therefore, hypothesized that loss of LKB1promotes an immune-suppressed phenotype in KRAS LUAD. To test this, we analyzed 100 immune-related genes from The Cancer Genome Atlas datasets, as well as three other sets of resected tumors from NSLC patients from MD Anderson Cancer Center. Results: Our results revealed that tumors lacking LKB1 (LKB1-loss), as defined by mutation or gene-expression signature, resulted in a down regulation of: tumor associated immunosuppressive ligands, PD-L1, PD-L2, B7-H3, B7-H4, HVEM; T cell associated immunosuppressive ligands PD-1, CTLA-4, BTLA; co-stimulation ligands such as CD80, -86, -40L, and -40. We further examined if this loss of immunosuppression and co-stimulation resulted in changes in markers of T cell phenotypes. Indeed, tumors lacking LKB1-loss tumors demonstrated a distinct lack absence of several T cell markers including CD3, -4, & -8, but also RORC, T-bet, GATA-3, and FoxP3. To determine if myeloid cell populations, known mediators of immune suppression, were also diminished in LKB1-loss tumors, we analyzed expression of CD11c, -103, -11b, -68, and CD33. Consistent with T cell makers and suppressive ligand markers, myeloid markers were down regulated in LKB1-loss tumors. Because common immune suppression markers were lost, we then examined the expression of antigen presentation (HLA-I, HLA-II) and amino acid transporters as possible alternative methods for immune suppression. In LKB1-loss tumors, both HLA-I and HLA-II markers were down regulated, compared to tumors with intact LKB1 expression. Furthermore, genes associated with the antigen presentation complex, LMP2, TAP1&2, and B2M were also down regulated. In contrast to the immune markers described above, cationic transporters (SLC7A2), anionic transporters (SLC7A11), and neutral transporters (SLC3A2) were universally upregulated in LKB1-loss tumors. Conclusions: LKB1 loss is associated with an inert immune phenotype. Our data suggests that reduced antigen presentation and amino acid depletion, but not checkpoint factors or MDSCs, are likely mediator of this phenotype. Citation Format: Warren L. Denning, Ferdinandos Skoulidis, Li Shen, Vassiliki Papadimitrakopoulou, Lixia Diao, Yanyan Lou, Lauren A. Byers, Jing Wang, Jaime R. Canales, Ignacio I. Wistuba, John Weinstein, Don L. Gibbons, John V. Heymach. Loss of LKB1 mediates an immune inert phenotype in human lung adenocarcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4137.

Abstract Introduction: A and T are associated with the development of cardiac dysfunction. The kinetics of cardiac biomarkers early after A or T treatment (Rx) is undefined. Methods: We studied hs-TnT (detection limit: 3 ng/ml, myocardial infarction threshold 14 ng/ml) and nt-proBNP (detection limit: 5 pg/ml) levels just prior to (baseline), and on days +1, +2, +3 and +7 during the first and second cycle in breast cancer pts treated with A (n = 11) or T (n = 11). Baseline and peak values for each cycle, baseline to baseline, peak to peak, time to peak, and baseline corrected area under the curve (AUC) were calculated. Study No. NCT01771549 Results: Group (grp) A pts were younger, 51 ± 18 years, than T grp pts, 60 ± 13 years. Three pts in A grp and 4 pts in T grp used antihypertensives. Baseline ejection fraction (EF) was 52-68% (A grp) and 56-69% (T grp). Hs-TnT was undetectable in 5/11 A grp, and 2/11 T grp pts at baseline. By cycle 2 day +1, all A pts had a detectable level of hs-TnT (p = 0.03) compared to 7/11 T pts. Median time to peak level for hs-TnT was 1.5 days for the A grp and 1.2 days for the T grp (NS). In the A grp, significant transient increase in nt-proBNP were also seen (Table). In the T grp, a significant rise in nt-proBNP was not seen in cycle 1 but was seen on cycle 2 day +1 and cycle 2 peak. AUC values were positive for hs-TnT in A pts but not in T pts, and were positive for nt-proBTNP for both A and T. Only 1 pt in A grp had symptomatic EF decline (47%) three months from start of A. None of T grp pts had EF decline. Conclusion: A, but not T was associated with increase in pre-Rx (cycle 2 only), peak, and AUC hs-TnT levels for cycles 1 and 2 with peak values occurring 1.5 days post-Rx. This study evaluating serial kinetics of cardiac biomarker in pts receiving A or T therapy, suggests that assessment of pre- and day +2 post-Rx values could be a means of quantifying cumulative myocardial injury over the chemotherapy course. *Roche Diagnostics Corp, Indianapolis, IN provided assay reagent kits. hs-TnT, ng/mlA, cycle 1A, cycle 2P-valT, cycle 1T, cycle 2P-valBaseline4.69.3<0.0027.75.0NSPeak9.6**16.1†<0.0028.06.8NSTime to peak, days1.61.5NS1.11.3NSAUC above baseline7.1±13.427±19<0.001-1.4±7.2-5±17NSnt-proBNP, pg/mlA, cycle 1A, cycle 2P-valT, cycle 1T, cycle 2P-valBaseline9490NS12480NSPeak361*359*NS209206†NSTime to peak, days1.81.9NS2.21.8NSAUC above baseline423499NS78**-32**<0.5*p<0.001,† p ≤0.01††p = 0.002NS = statistically non-significant (p>0.05)**p<0.03 Citation Format: Pooja Advani, Jonathan Hoyne, Alvaro Moreno-Aspitia, Marcia Dubin, Shelly Brock, Caroline Harlow, Tammy Wollett, Saranya Chumsri, Joseph Blackshear. A prospective pilot study of kinetics of high sensitivity troponin T (hs-TnT) and amino terminal (nt-proBNP) in breast cancer patients (pts) treated with doxorubicin (A) or trastuzumab (T). [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3930.