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Background Significant clinical and research applications are driving large scale adoption of individualized tumor sequencing in cancer in order to identify tumors-specific mutations. When a matched germline sample is available, somatic mutations may be identified using comparative callers. However, matched germline samples are frequently not available such as with archival tissues, which makes it difficult to distinguish somatic from germline variants. While population databases may be used to filter out known germline variants, recent studies have shown private germline variants result in an inflated false positive rate in unmatched tumor samples, and the number germline false positives in an individual may be related to ancestry. Methods First, we examined the relationship between the germline false positives and ancestry. Then we developed and implemented a tumor only caller (LumosVar) that leverages differences in allelic frequency between somatic and germline variants in impure tumors. We used simulated data to systematically examine how copy number alterations, tumor purity, and sequencing depth should affect the sensitivity of our caller. Finally, we evaluated the caller on real data. Results We find the germline false-positive rate is significantly higher for individuals of non-European Ancestry largely due to the limited diversity in public polymorphism databases and due to population-specific characteristics such as admixture or recent expansions. Our Bayesian tumor only caller (LumosVar) is able to greatly reduce false positives from private germline variants, and our sensitivity is similar to predictions based on simulated data. Conclusions Taken together, our results suggest that studies of individuals of non-European ancestry would most benefit from our approach. However, high sensitivity requires sufficiently impure tumors and adequate sequencing depth. Even in impure tumors, there are copy number alterations that result in germline and somatic variants having similar allele frequencies, limiting the sensitivity of the approach. We believe our approach could greatly improve the analysis of archival samples in a research setting where the normal is not available. Electronic supplementary material The online version of this article (10.1186/s12920-017-0296-8) contains supplementary material, which is available to authorized users.

Background Primary cardiac angiosarcomas are rare, but they are the most aggressive type of primary cardiac neoplasms. When patients do present, it is with advanced pulmonary and/or cardiac symptoms. Therefore, many times the correct diagnosis is not made at the time of initial presentation. These patients have metastatic disease and the vast majority of these patients die within a few months after diagnosis. Currently the treatment choices are limited and there are no targeted therapies available. Case presentation A 56-year-old male presented with shortness of breath, night sweats, and productive cough for a month. Workup revealed pericardial effusion and multiple bilateral pulmonary nodules suspicious for metastatic disease. Transthoracic echocardiogram showed a large pericardial effusion and a large mass in the base of the right atrium. Results of biopsy of bilateral lung nodules established a diagnosis of primary cardiac angiosarcoma. Aggressive pulmonary disease caused rapid deterioration; the patient went on hospice and subsequently died. Whole exome sequencing of the patient’s postmortem tumor revealed a novel KDR (G681R) mutation, and focal high-level amplification at chromosome 1q encompassing MDM4, a negative regulator of TP53. Conclusion Mutations in KDR have been reported previously in angiosarcomas. Previous studies also demonstrated that KDR mutants with constitutive KDR activation could be inhibited with specific KDR inhibitors in vitro. Thus, patients harboring activating KDR mutations could be candidates for treatment with KDR-specific inhibitors. Electronic supplementary material The online version of this article (doi:10.1186/s12885-016-3000-z) contains supplementary material, which is available to authorized users.

Femtosecond X-ray crystallography allows structural analysis of a difficult-to-crystallize fusion protein that is a potential component of a candidate HIV-1 subunit vaccine., CTB-MPR is a fusion protein between the B subunit of cholera toxin (CTB) and the membrane-proximal region of gp41 (MPR), the transmembrane envelope protein of Human immunodeficiency virus 1 (HIV-1), and has previously been shown to induce the production of anti-HIV-1 antibodies with antiviral functions. To further improve the design of this candidate vaccine, X-ray crystallography experiments were performed to obtain structural information about this fusion protein. Several variants of CTB-MPR were designed, constructed and recombinantly expressed in Escherichia coli. The first variant contained a flexible GPGP linker between CTB and MPR, and yielded crystals that diffracted to a resolution of 2.3 Å, but only the CTB region was detected in the electron-density map. A second variant, in which the CTB was directly attached to MPR, was shown to destabilize pentamer formation. A third construct containing a polyalanine linker between CTB and MPR proved to stabilize the pentameric form of the protein during purification. The purification procedure was shown to produce a homogeneously pure and monodisperse sample for crystallization. Initial crystallization experiments led to pseudo-crystals which were ordered in only two dimensions and were disordered in the third dimension. Nanocrystals obtained using the same precipitant showed promising X-ray diffraction to 5 Å resolution in femtosecond nanocrystallography experiments at the Linac Coherent Light Source at the SLAC National Accelerator Laboratory. The results demonstrate the utility of femtosecond X-ray crystallography to enable structural analysis based on nano/microcrystals of a protein for which no macroscopic crystals ordered in three dimensions have been observed before.

DNA focused panel sequencing has been rapidly adopted to assess therapeutic targets in advanced/refractory cancer. Integrated Genomic Profiling (IGP) utilising DNA/RNA with tumour/normal comparisons in a Clinical Laboratory Improvement Amendments (CLIA) compliant setting enables a single assay to provide: therapeutic target prioritisation, novel target discovery/application and comprehensive germline assessment. A prospective study in 35 advanced/refractory cancer patients was conducted using CLIA-compliant IGP. Feasibility was assessed by estimating time to results (TTR), prioritising/assigning putative therapeutic targets, assessing drug access, ascertaining germline alterations, and assessing patient preferences/perspectives on data use/reporting. Therapeutic targets were identified using biointelligence/pathway analyses and interpreted by a Genomic Tumour Board. Seventy-five percent of cases harboured 1–3 therapeutically targetable mutations/case (median 79 mutations of potential functional significance/case). Median time to CLIA-validated results was 116 days with CLIA-validation of targets achieved in 21/22 patients. IGP directed treatment was instituted in 13 patients utilising on/off label FDA approved drugs (n = 9), clinical trials (n = 3) and single patient IND (n = 1). Preliminary clinical efficacy was noted in five patients (two partial response, three stable disease). Although barriers to broader application exist, including the need for wider availability of therapies, IGP in a CLIA-framework is feasible and valuable in selection/prioritisation of anti-cancer therapeutic targets.

Glassy cell carcinoma of the cervix (GCCC) is a very rare and aggressive form of cervical cancer. An adolescent female with advanced metastatic disease was enrolled in our genomic profiling research protocol. We identified high-level amplification of epidermal growth factor receptor (EGFR) and Yes-associated protein-1 (YAP1), which led to the addition of EGFR inhibitors to the chemotherapy regimen. Here, we report the first genetically profiled case of GCCC with potential therapeutic implications.

miRNAs play an important role in the regulation of a wide assortment of cellular processes by sequestering target mRNAs and inhibiting translation of the proteins that they encode. Multiple miRNAs can regulate single mRNA molecules and, alternatively, a single miRNA can act on a number of mRNA targets. Dysfunctional miRNAs are commonly found in a variety of solid cancers and are attractive candidates for next-generation therapeutics. This article highlights miRNA signatures proposed for lung cancer classification and diagnosis, chemo- and radio-therapy resistance, metastasis and prediction of treatment outcome and survival.

Tyrosine kinase inhibitors (TKIs) have been in use as cancer therapeutics for nearly a decade, and their utility in targeting specific malignancies with defined genetic lesions has proven to be remarkably effective. Recent efforts to characterize the spectrum of genetic lesions found in non-small cell lung carcinoma (NSCLC) have provided important insights into the molecular basis of this disease and have also revealed a wide array of tyrosine kinases that might be effectively targeted for rationally designed therapies. The findings of these studies, however, also provide a cautionary tale about the limitations of single-agent therapies, which fail to account for the genetic heterogeneity and pathway redundancy that characterize advanced NSCLC. Emergence of drug resistance mechanisms to specific TKIs, such as gefitinib and erlotinib, suggests that more sophisticated chemotherapeutic paradigms that target multiple pathways at the same time will be required to effectively treat this disease.

Organophosphorous pesticides and nerve agents inhibit the enzyme acetylcholinesterase at neuronal synapses and in neuromuscular junctions. The resulting accumulation of acetylcholine overwhelms regulatory mechanisms, potentially leading to seizures and death from respiratory collapse. While current therapies are only capable of reducing mortality, elevation of the serum levels of the related enzyme butyrylcholinesterase (BChE) by application of the purified protein as a bioscavenger of organophosphorous compounds is effective in preventing all symptoms associated with poisoning by these toxins. However, BChE therapy requires large quantities of enzyme that can easily overwhelm current sources. Here, we report genetic optimization, cloning and high-level expression of human BChE in plants. Plant-derived BChE is shown to be biochemically similar to human plasma-derived BChE in terms of catalytic activity and inhibitor binding. We further demonstrate the ability of the plant-derived bioscavenger to protect animals against an organophosphorous pesticide challenge.

Plants are potentially the most economical platforms for the large-scale production of recombinant proteins. Thus, plant-based expression of subunit human immunodeficiency virus type 1 (HIV-1) vaccines provides an opportunity for their global use against the acquired immunodeficiency syndrome pandemic. CTB-MPR(649-684)[CTB, cholera toxin B subunit; MPR, membrane proximal (ectodomain) region of gp41] is an HIV-1 vaccine candidate that has been shown previously to induce antibodies that block a pathway of HIV-1 mucosal transmission. In this article, the molecular characterization of CTB-MPR(649-684) expressed in transgenic Nicotiana benthamiana plants is reported. Virtually all of the CTB-MPR(649-684) proteins expressed in the selected line were shown to have assembled into pentameric, GM1 ganglioside-binding complexes. Detailed biochemical analyses on the purified protein revealed that it was N-glycosylated, predominantly with high-mannose-type glycans (more than 75%), as predicted from a consensus asparagine-X-serine/threonine (Asn-X-Ser/Thr) N-glycosylation sequon on the CTB domain and an endoplasmic reticulum retention signal attached at the C-terminus of the fusion protein. Despite this modification, the plant-expressed protein retained the nanomolar affinity to GM1 ganglioside and the critical antigenicity of the MPR(649-684) moiety. Furthermore, the protein induced mucosal and serum anti-MPR(649-684) antibodies in mice after mucosal prime-systemic boost immunization. Our data indicate that plant-based expression can be a viable alternative for the production of this subunit HIV-1 vaccine candidate.

Therapeutically valuable proteins are often rare and/or unstable in their natural context, calling for production solutions in heterologous systems. A relevant example is that of the stress-induced, normally rare, and naturally unstable "read-through" human acetylcholinesterase variant, AChE-R. AChE-R shares its active site with the synaptic AChE-S variant, which is the target of poisonous organophosphate anticholinesterase insecticides such as the parathion metabolite paraoxon. Inherent AChE-R overproduction under organophosphate intoxication confers both short-term protection (as a bioscavenger) and long-term neuromuscular damages (as a regulator). Here we report the purification, characterization, and testing of human, endoplasmic reticulum-retained AChE-R(ER) produced from plant-optimized cDNA in Nicotiana benthamiana plants. AChE-R(ER) purified to homogeneity showed indistinguishable biochemical properties, with IC50 = 10(-7) M for the organophosphate paraoxon, similar to mammalian cell culture-derived AChE. In vivo titration showed dose-dependent protection by intravenously injected AChE-R(ER) of FVB/N male mice challenged with a lethal dose of paraoxon, with complete elimination of short-term clinical symptoms at near molar equivalence. By 10 days postexposure, AChE-R prophylaxis markedly limited postexposure increases in plasma murine AChE-R levels while minimizing the organophosphate-induced neuromuscular junction dismorphology. Our findings present plant-produced AChE-R(ER) as a bimodal agent, conferring both short- and long-term protection from organophosphate intoxication.

Nicotiana benthamiana plants were engineered to express a codon-optimized gene encoding the human acetylcholinesterase-R (AChE) isoform. The transgenic plants expressed the protein at >0.4% of total soluble protein, and the plant-produced enzyme was purified to homogeneity. Following lysis, procainamide affinity chromatography and anion-exchange chromatography, more than 400-fold purification was achieved and electrophoretic purity was obtained. This pure protein is kinetically indistinguishable from the only commercially available source of human acetylcholinesterase, which is produced in mammalian cell culture. Thus, we have demonstrated a model system for the production of acetylcholinesterase, which is not susceptible to the quantitative limitations or mammalian pathogens associated with purification from mammalian cell culture or human serum.

Background. Treatment of triple-negative breast cancer (TNBC) is clinically challenging due to disease heterogeneity and aggressiveness which often result in high recurrence and mortality rates. We have previously reported on genomic alterations found in 14 metastatic TNBC tumors (Craig et al 2012) uncovered through whole-genome and transcriptome sequencing and found that majority of tumor changes in our cohort converged on RAS/RAF/MEK/ERK and PI3K/AKT/mTOR pathways revealing potential therapeutic targets. In this study, we interrogated patients’ matched primary and metastatic cancers using exome sequencing. Differentiating between primary and metastasis-specific genomic alterations will further our understanding of TNBC recurrence. Methods. Tumor tissue was microdissected from 10uM FFPE sections from primary tumors of nine TNBC patients. DNA was extracted using Qiagen AllPrep kit. Whole-genome libraries were prepared from >250ng of DNA using Kapa-on–Bead method. Exomes were captured using All Exon V5 capture kit (Agilent Technologies) and sequenced on Illumina HiSeq. List of somatic variants (SNVs) in primary tissue was generated using Seurat variant caller and manually compared with the SNVs uncovered previously in metastatic tumors. Presence (or absence) of all reported somatic SNVs was visually confirmed in IGV genome viewer. Results. Our analysis revealed the majority of somatic mutations found in metastatic lesions were also present in the primary tissue (64.3% on average). Patient TNBC03 had an unusually high number of new mutations in the lung metastatic lesion (72.7%). All TP53 mutations in our cohort were an early event present in both primary and metastatic tissues. In patient TNBC01, acquired mutation in NF1 - an upstream negative regulator of RAS pathway - in addition to pre-existing PTEN deletion, CTNNA1 underexpression and RB1 mutation - suggests RAS pathway addiction which could have been responsible for disease recurrence in this patient. Interestingly, NEDD4 mutation was found in a lung metastasis of another patient whose disease was refractory to BEZ235 (PI3K/mTOR inhibitor) treatment. NEDD4 has recently been shown to be an upstream regulator of the PI3K pathway and a possible therapeutic target in cancers with downregulated PTEN. Additional select acquired mutations in metastatic lesions of our cohort included FGF14, ABCB10, MDM2, KIF1C, ATAD2B, PTPLAD2, MDM20, TDRD1, and USP6. Mutated CDH5 was found in metastases in 2 of 9 patients. Conclusion. Our findings demonstrate the complexity and variability in somatic events underlying TNBC recurrence, reinforcing the need to re-biopsy metastatic TNBC lesions and to employ combination targeted therapies wherever possible. Citation Format: Irene Cherni, Maren K Levin, Joyce A O"Shaughnessy, John D Carpten. Comparative analysis of somatic mutations in matched primary vs metastatic triple-negative breast cancers [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P2-03-04.

Background: The promise of precision medicine is vast and efforts are needed to increase utilization of precision oncology tests. Studies have suggested that mutations in cancer predisposition are more common than previously thought. Herein, we investigated germline alterations in cancer patients undergoing tumor profiling to identify the potential genetic predisposing factors as well as therapeutically relevant germline variants. Methods: Comprehensive genomic profiling was performed on tumor samples from 67 patients from 24 cancer types with ages ranging 23-77 years. We performed targeted exome sequencing of 562 genes in tumor and paired normal DNA which included 116 genes that have been associated with cancer predisposition syndromes. In addition to identifying the somatic changes, we characterized germline mutations in select 116 genes using public databases (ClinVar, Ensembl). Results: Pathogenic germline mutations were found in 17.91% (12/67) of cases, which included 10 unique variants in 8 genes, the majority of which included DNA repair genes (ATM, BRCA2, ERCC4, NBN, PSM2, MUTYH, XPC and AR). Likely pathogenic mutations were found in 23% (16/67) of the cases and 12% (8/67) were predicted pathogenic by SIFT/PolyPhen. In select cases we found existence of a 2nd hit which may have an impact on certain targeted therapeutics such as PARP inhibitors. All of the cases profiled carried either benign or variants of unknown significance (VUS) mutations. Germline findings in cancer susceptibility genes that were concordant with the individual's cancer type included ATM (R2227C) in HER2+ breast cancer patient; BRCA2 missense mutation (K944X) in peritoneal cancer and HER2+ breast cancer; BRCA2 (W1692MfsX3, I2588YfsX60) in patients with pancreatic and breast cancer respectively and PMS2(L729QfsX6) in colorectal cancer (CRC). Germline pathogenic findings in cancer susceptibility genes that were dis-concordant with the individual's cancer type included ERCC4 (R799W) in endometrial cancer; XPC (P334H) in CRC; MUTYH (G393D) in esophageal and CRC; NBN (K219NfsX16) in HER2+ breast cancer and AR (A646D) mutation in CRC. Interestingly, FLT4 (R1324L) mutation which is associated with decreased response and toxicity to sunitinib in renal cell cancer was found across several tumor types including lung adenocarcinoma (n = 3), pancreatic cancer (n = 3), HER2+ breast cancer (n = 2), CRC (n = 2) and n = 1 in gallbladder carcinoma, cholangiocarcinoma and endometrial adenocarcinoma. The comparison of somatic to germline mutation will be presented. Conclusion: Germline data analysis of a small cohort of advanced cancer patients revealed that unselected cancer patients may benefit from germline evaluation which may influence the evaluation and care of the patient or the patient's family members. Even though majority of germline mutations were VUS, several mutations were identified which may be amenable to targeted therapeutic strategies. Citation Format: Subha Krishnan, Gargi D. Basu, Laura Gonzalez-Malerva, Thomas Royce, Tracey White, Janine R. Lobello, Amy King, Kevin Lau, Ahmet Kurdoglu, Matthew Halbert, Jeff Kiefer, Irene Cherni, Jessica Aldrich, Tyler Izatt, Megan Russell, Sara Nasser, John D. Carpten, David W. Craig, Jeffrey Trent, Michael J. Demeure. Germline findings in targeted tumor sequencing using matched normal DNA. [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 4493.

671 Background: The gene encoding the AT-rich interacting domain (ARID1A) is a tumor suppressor and a subunit of the Switch/Sucrose Non fermentable (SWI/SNF) chromatin remodeling complex that regulates gene expression. Additionally, ARID1A functions to maintain genomic integrity by interacting with ATR. Mutations in ARID1A have been observed in a number of cancers. Inactivation of ARID1A has been reported to lead to increased sensitivity to inhibitors of PARP and PI3K/AKT pathway. We evaluated ARID1A alterations in gastrointestinal (GI) cancers to identify new therapeutic options. Methods: Comprehensive genomic profiling was performed on 55 GI cancer samples with 28 metastatic and 27 primary samples. Targeted sequencing was performed on 562 cancer associated genes in paired tumor and blood DNA samples. Results: ARID1A alterations were noted in 20% (11/55) of GI cancers. Alterations included frameshift and nonsense mutations, INDELs and structural breakpoints which are predicted to cause loss of ARID1A function. Tumor tissues harboring ARID1A alterations included 2/8 gastro-esophageal cancers, 2/2 duodenal cancers, 3/29 colorectal (CRC) cancers, 2/2 small bowel cancers, and 2/7 bile duct cancers. Mutations in the C-terminal half of ARID1A, known to interact with ATR and mediate its recruitment to double strand breakpoints was present in 2/11 samples. No pathogenic changes in the DNA repair genes (BRCA1/2, CHEK1/2, ATM, ATR or PALB2) or MMR genes were present in the tumors harboring ARID1A alterations. Interestingly, ARID1A mutations frequently coexisted with alterations leading to activation of the PI3K pathway. We found 45% (5/11) samples with mutated ARID1A had pathogenic alterations in PIK3CA, AKT, NF1, STK11, TSC1 and TSC2 genes which may increase sensitivity to PI3K pathway inhibitors. Conclusions: Targeted sequencing demonstrated that ARID1A is a frequent event in GI cancer. Our results suggest there may be a correlation between samples with ARID1A alteration and PI3K/AKT pathway activation which may lead to therapeutic opportunities with PI3K pathway inhibitors. The presence of ARID1A mutations especially those in the C-terminal half may predict clinical utility of PARP inhibitors in GI cancer.

Background Maxillary sinus carcinoma (MSC) is a rare cancer of the head and neck region. Patients are treated with surgery, radiation therapy, and chemotherapy and the treatment regimen is based on patient’s age, general health condition, disease stage, and its extent of spread. There is very little information available on the genetics of this disease. DNA content based flow sorting of tumor cells followed by array comparative genomic hybridization allows for high definition global assessment of distinct clonal changes within tumor populations. Methods We applied this technique to primary and metastatic samples collected from a patient with radio- and chemotherapy refractory maxillary sinus carcinoma to gauge the progression of this disease. Results A clonal KIT amplicon was present in aneuploid populations sorted from the primary tumor and in divergent subclones arising in metastatic foci found in the brain, lung, and jejunum. The evolution of these subclones was associated with distinct genetic aberrations and DNA ploidies. Conclusion The information presented here paves the path to understanding the development and progression of this disease.

Previous studies have now demonstrated that both genic and global hypomethylation characterizes the multiple myeloma (MM) epigenome. Whether these methylation changes are associated with global and corresponding increases (or decreases) in transcriptional activity are poorly understood. The purpose of our current study was to correlate DNA methylation levels in MM to gene expression. We analyzed matching datasets generated by the GoldenGate methylation BeadArray and Affymetrix gene expression platforms in 193 MM samples. We subsequently utilized two independent statistical approaches to identify methylation-expression correlations. In the first approach, we used a linear correlation parameter by computing a Pearson correlation coefficient. In the second approach, we discretized samples into low and high methylation groups and then compared the gene expression differences between the groups. Only methylation of 2.1% and 25.3% of CpG sites on the methylation array correlated to gene expression by Pearson correlation or the discretization method, respectively. Among the genes with methylation-expression correlations were IGF1R, DLC1, p16, and IL17RB. In conclusion, DNA methylation may directly regulate relatively few genes and suggests that additional studies are needed to determine the effects of genome-wide methylation changes in MM.

Background Olfactory neuroblastoma (ONB) is a rare cancer of the sinonasal tract with little molecular characterization. We performed whole genome sequencing (WGS) on paired normal and tumor DNA from a patient with metastatic-ONB to identify the somatic alterations that might be drivers of tumorigenesis and/or metastatic progression. Methodology/Principal Findings Genomic DNA was isolated from fresh frozen tissue from a metastatic lesion and whole blood, followed by WGS at >30X depth, alignment and mapping, and mutation analyses. Sanger sequencing was used to confirm selected mutations. Sixty-two somatic short nucleotide variants (SNVs) and five deletions were identified inside coding regions, each causing a non-synonymous DNA sequence change. We selected seven SNVs and validated them by Sanger sequencing. In the metastatic ONB samples collected several months prior to WGS, all seven mutations were present. However, in the original surgical resection specimen (prior to evidence of metastatic disease), mutations in KDR, MYC, SIN3B, and NLRC4 genes were not present, suggesting that these were acquired with disease progression and/or as a result of post-treatment effects. Conclusions/Significance This work provides insight into the evolution of ONB cancer cells and provides a window into the more complex factors, including tumor clonality and multiple driver mutations.

Constitutive activation of STAT3 is a common feature in many solid tumors including non-small cell lung carcinoma (NSCLC). While activation of STAT3 is commonly achieved by somatic mutations to JAK2 in hematologic malignancies, similar mutations are not often found in solid tumors. Previous work has instead suggested that STAT3 activation in solid tumors is more commonly induced by hyperactive growth factor receptors or autocrine cytokine signaling. The interplay between STAT3 activation and other well-characterized oncogenic “driver” mutations in NSCLC has not been fully characterized, though constitutive STAT3 activation has been proposed to play an important role in resistance to various small-molecule therapies that target these oncogenes. In this study we demonstrate that STAT3 is constitutively activated in human NSCLC samples and in a variety of NSCLC lines independent of activating KRAS or tyrosine kinase mutations. We further show that genetic or pharmacologic inhibition of the gp130/JAK2 signaling pathway disrupts activation of STAT3. Interestingly, treatment of NSCLC cells with the JAK1/2 inhibitor ruxolitinib has no effect on cell proliferation and viability in two-dimensional culture, but inhibits growth in soft agar and xenograft assays. These data demonstrate that JAK2/STAT3 signaling operates independent of known driver mutations in NSCLC and plays critical roles in tumor cell behavior that may not be effectively inhibited by drugs that selectively target these driver mutations.

Background Recent advances in the treatment of cancer have focused on targeting genomic aberrations with selective therapeutic agents. In rare tumors, where large-scale clinical trials are daunting, this targeted genomic approach offers a new perspective and hope for improved treatments. Cancers of the ampulla of Vater are rare tumors that comprise only about 0.2% of gastrointestinal cancers. Consequently, they are often treated as either distal common bile duct or pancreatic cancers. Methods We analyzed DNA from a resected cancer of the ampulla of Vater and whole blood DNA from a 63 year-old man who underwent a pancreaticoduodenectomy by whole genome sequencing, achieving 37× and 40× coverage, respectively. We determined somatic mutations and structural alterations. Results We identified relevant aberrations, including deleterious mutations of KRAS and SMAD4 as well as a homozygous focal deletion of the PTEN tumor suppressor gene. These findings suggest that these tumors have a distinct oncogenesis from either common bile duct cancer or pancreatic cancer. Furthermore, this combination of genomic aberrations suggests a therapeutic context for dual mTOR/PI3K inhibition. Conclusions Whole genome sequencing can elucidate an oncogenic context and expose potential therapeutic vulnerabilities in rare cancers.

Introduction The Hedgehog Signaling Pathway (HHSP) has been implicated in the development of multiple cancers. HHSP activation may primarily be hedgehog ligand-dependent in non-small cell lung cancer (NSCLC); while a subset may be ligand-independent. In this study NSCLC primary tumors were used to identify correlations between multiple biomarkers thought to be involved in the HHSP and the clinical outcomes of patients with NSCLC. Identification of such correlations could be used to aid in NSCLC treatment and predicting patient prognosis. Methods A tissue microarray representing 248 clinically annotated stage I–II NSCLC cases was stained using immunohistochemistry (IHC) and scored for HHSP proteins namely, SHH, PTCH1, SMO, GLI1, and GLI2; as well as, ALDH1A1, a putative cancer stem cell marker. Data was analyzed for correlation between IHC staining, EGFR and KRAS mutations, and clinical characteristics including relapse-free survival (RFS) and overall survival (OS). Results In adenocarcinoma, there were significant correlations of IHC expression between SHH and downstream HHSP receptor SMO ( p =0.017) and transcription factor GLI1 ( p =0.001), while SMO correlated with GLI1 ( p =0.007). In squamous cell carcinoma, SHH significantly correlated with GLI2 protein expression ( p =0.003). After multiple testing correction, there was no significant correlation between any of the six markers and RFS or OS. Conclusions Key downstream components of the HHSP show correlation with sonic hedgehog ligand (SHH) expression, suggesting that ligand-dependent signaling is more prevalent in primary NSCLC tumors. Surprisingly, in early-stage NSCLC, there were no significant correlations between HHSP proteins or ALDH1A1 and RFS or OS.

The concept of using cholinesterase bioscavengers for prophylaxis against organophosphorous nerve agents and pesticides has progressed from the bench to clinical trial. However, the supply of the native human proteins is either limited (e.g., plasma-derived butyrylcholinesterase and erythrocytic acetylcholinesterase) or nonexisting (synaptic acetylcholinesterase). Here we identify a unique form of recombinant human butyrylcholinesterase that mimics the native enzyme assembly into tetramers; this form provides extended effective pharmacokinetics that is significantly enhanced by polyethylene glycol conjugation. We further demonstrate that this enzyme (but not a G117H/E197Q organophosphorus acid anhydride hydrolase catalytic variant) can prevent morbidity and mortality associated with organophosphorous nerve agent and pesticide exposure of animal subjects of two model species.

Plants are potentially the most economical platforms for the large-scale production of recombinant proteins. Thus, plant-based expression of subunit human immunodeficiency virus type 1 (HIV-1) vaccines provides an opportunity for their global use against the acquired immunodeficiency syndrome pandemic. CTB-MPR(649-684)[CTB, cholera toxin B subunit; MPR, membrane proximal (ectodomain) region of gp41] is an HIV-1 vaccine candidate that has been shown previously to induce antibodies that block a pathway of HIV-1 mucosal transmission. In this article, the molecular characterization of CTB-MPR(649-684) expressed in transgenic Nicotiana benthamiana plants is reported. Virtually all of the CTB-MPR(649-684) proteins expressed in the selected line were shown to have assembled into pentameric, GM1 ganglioside-binding complexes. Detailed biochemical analyses on the purified protein revealed that it was N-glycosylated, predominantly with high-mannose-type glycans (more than 75%), as predicted from a consensus asparagine-X-serine/threonine (Asn-X-Ser/Thr) N-glycosylation sequon on the CTB domain and an endoplasmic reticulum retention signal attached at the C-terminus of the fusion protein. Despite this modification, the plant-expressed protein retained the nanomolar affinity to GM1 ganglioside and the critical antigenicity of the MPR(649-684) moiety. Furthermore, the protein induced mucosal and serum anti-MPR(649-684) antibodies in mice after mucosal prime-systemic boost immunization. Our data indicate that plant-based expression can be a viable alternative for the production of this subunit HIV-1 vaccine candidate.

156 Background: Whether EGFR is a critical target in met TNBC is unknown. Here we report the clinical history & tumor molecular alterations in a patient with refractory metTNBC who had an ExRx to pac/cis. Methods: Following IRB-approved informed consent, targeted NGS (Foundation Medicine) and WGS (TGEN) was performed on the pt’s FFPE primary TNBC and 2 recurrent lymph nodes to characterize all classes of genomic alterations in cancer-related genes. RPPA was performed at a CLIA-certified laboratory (Theranostics Health) and George Mason Univ where immunostaining was directed against HER1/2/3 pathway and other proteins. Results: At age 58 in 2006, pt had T1c 1+ node TNBC treated with FAC/T. Between 2008 and 2011 she had 4 chemotherapy-refractory recurrences in axilla, supraclavicular (SC), internal mammary (IM) LNs treated unsuccessfully with surgery, radiation and multiple cytotoxic agents including carboplatin. In 2011, following SC LN biopsy, she was treated with BEZ, a PI3K/mTOR, ATM, ATR, DNA-PKcs inhibitor, had a 3 mo response, followed by rapidly enlarging progressive disease (PD) in IM LNs pushing sternum anteriorly. She was treated with pac/cis and had an ongoing complete response (CR) of 2.5+ yrs. NGS of 2011 SC LN (pre-BEZ) & 2012 IM LN (post-BEZ): TP53 & BRCA2 (somatic 15% mutant allele freq) mutations, FOXM1 amplification; SMARCA4 (BRG1) deletion RPPA (GMU) 2011 SC LN (Pre-BEZ): 3+ EGFR; 2+ p-EGFR, p-AKT, p-MEK1/2, p-mTOR RPPA (Theranostics) 2012 IM LN (post-BEZ): 3+ p-MEK1/2 (EGFR & p-EGFR 0) (AR-). Conclusions: Strong EGFR signaling associated with chemo-resistant metTNBC in 2011 SC LN was not present in post-BEZ rapid PD in IM LN which then had durable CR with pac/cis. BEZ inhibits DSB repair, sensitizing cancers to DNA damaging agents (Gil del Alcazar, Clin Ca Res 20:1235, 2014). Progression of p-AKT-activated TNBC following response to inhibitors of PI3K & DNA repair shows DSB repair-deficiency and MAPK activation (Juvekar, Cancer Dis 2:1048, 2012). A prospective trial of BEZ followed at PD by pac/cis in metTNBC is warranted.

126 Background: Breast cancer is a molecularly diverse disease with molecular subtypes defined by ER, PR and HER2 status. Dysregulation of the cell cycle pathway through activation of cyclin D1 and/or inactivation of CDKN2A is a mechanism of tumor progression in breast cancer and drugs targeting CDK4/6 is undergoing investigation in this disease with promising preliminary results. We investigated several key genetic alterations in cell cycle regulatory proteins in advanced breast cancer patient samples to identify potential targets for cell cycle related treatment options. Methods: A comprehensive genomic profiling was performed on 37 breast cancer samples with the majority being metastatic. Testing included targeted exome sequencing of 562 genes in tumor and paired normal DNA. Results: Patients’ ages ranged from 27-66 years. Analysis of cell cycle regulatory genes revealed focal amplification of cyclin D1/D2 or CDKN2A loss in 9/37 (24%) of cases, of which, 45% were hormone receptor (HR) positive, HER2 negative, 33% were HR positive, HER2 positive and 22% were triple negative (TNBC) subtype. Loss of RB1 was analyzed since it negatively regulates response to CDK4/6 inhibitors. Loss of RB1 was noted in 4/37 (10.8%) of cases, all of which were TNBCs. Only a single TNBC with RB1 loss had concurrent CDK4 amplification and loss of CDKN2A. No evidence of CCNE1 amplification was found in any samples. Incidence of CDK4 and CDK6 amplification was found in samples with cyclin D1 amplification or CDKN2A loss. Our cohort included a single male TNBC patient who harbored cyclin D1 amplification as well as CDKN2A loss. Overall, amplification of cyclin D1, cyclin D2, CDK4 and CDK6 was found in 16%, 5.4%, 2.7% and 2.7% respectively. Loss of CDKN2A was noted in 5.4% of tumors profiled. Conclusions: Exome sequencing identified subsets of patients with mutated cell cycle associated genes that may benefit from CDK 4/6 inhibitors. Our data suggests loss of RB1 is more frequent in TNBC subtype which may confer resistance to CDK4/6 inhibitors. Our analysis suggests that in addition to HR positive, HER2 negative patients, a subset of HR positive, HER2 positive patients could be sensitive to CDK4/6 inhibitors due to activation of the cell cycle pathway as evidenced by mutations in key genes.

e12073 Background: We have characterized the genomic alterations (GAs) and phosphoproteome of matched primary and metTNBCs from AA women. Methods: Microdissected fresh metTNBCs (N = 6) were analyze...

As next-generation sequencing continues to have an expanding presence in the clinic, the identification of the most cost-effective and robust strategy for identifying copy number changes and translocations in tumor genomes is needed. We hypothesized that performing shallow whole genome sequencing (WGS) of 900-1000-bp inserts (long insert WGS, LI-WGS) improves our ability to detect these events, compared with shallow WGS of 300-400-bp inserts. A priori analyses show that LI-WGS requires less sequencing compared with short insert WGS to achieve a target physical coverage, and that LI-WGS requires less sequence coverage to detect a heterozygous event with a power of 0.99. We thus developed an LI-WGS library preparation protocol based off of Illumina's WGS library preparation protocol and illustrate the feasibility of performing LI-WGS. We additionally applied LI-WGS to three separate tumor/normal DNA pairs collected from patients diagnosed with different cancers to demonstrate our application of LI-WGS on actual patient samples for identification of somatic copy number alterations and translocations. With the evolution of sequencing technologies and bioinformatics analyses, we show that modifications to current approaches may improve our ability to interrogate cancer genomes.

Background: Cigarettes, food, water, and air pollution can contain toxic heavy metals, such as cadmium (Cd), arsenic (As), mercury (Hg), and lead (Pb). A cigarette contains approximately 1-2 ug of Cd, 40-120 ng of As, 2.95-10.2 ng of Hg, and 1-2 mg/kg of Pb because the tobacco plant concentrates metals from soil and water. The accumulation of toxic heavy metals inhaled from smoking may be associated with lung cancer. We surveyed tissue from current/former smokers with non-small cell lung cancer (NSCLC) for heavy metal content. Methods: Tissue collected from treatment-naïve stage I and II NSCLC patient samples that underwent surgical resection was analyzed by inductively coupled plasma mass spectroscopy (ICP-MS) for concentration of Cd, As, Hg, and Pb. 16 cases were matched for stage, histology, gender, age, and race. Concentrations were measured from two 20 micron thick slides each from formalin-fixed, paraffin-embedded (FFPE) tissues: primary lung cancer, benign adjacent lung, and where applicable to malignant lymph node, benign adjacent lymph node, benign lymph node, and distant non-malignant lung tissue. Negative controls were paraffin without tissue from FFPE blocks. Paired samples t-tests were used to make intra-case comparisons. The nonparametric Mann-Whitney U and the Wilcoxon signed-rank test were used to examine inter-case differences. Results: The median patient age was 64.7 (range 36.4-75.4). There were 15 primary lung cancer cases with sufficient tissue for analysis: 8 were men, 7 were stage I, 7 had adenocarcinoma, 8 had squamous cell carcinoma, 8 were former smokers, 7 were current smokers, 9 were Caucasian, and 6 were African-American. A total of 75 tissue samples were analyzed with an average sample weight of 2.9 mg. With the lower limit of detection at 0.001 ug/g, the median concentrations in ug/g for Cd, As, Hg, and Pb in primary lung tumor were 0.058 (range undetectable [Not]-0.316), 0.019 (range Not-0.078), 0 (range Not-0.015), and 0 (range Not-0.026); respectively. In distant non-malignant lung samples, the median concentrations in ug/g for Cd, As, Hg, and Pb were 0.204 (range Not-2.500), 0.025 (range Not-0.070), 0 (range Not-0.012), and Not; respectively. There were no significant inter-case differences for the heavy metal concentrations in primary lung tumor and stage, histology, gender, and race. However, distant non-malignant lung samples had significantly higher Cd levels compared to primary lung tumor (p=0.013). Conclusion: Using ICP-MS, concentrations of Cd, As, Hg, and Pb could be measured in low quantities of FFPE tissue. Significantly higher Cd content was observed in distant non-malignant lung tissue compared to primary lung tumor. There appears to be higher levels of Cd and As than Hg and Pb in the lung tissues of current/former smokers with NSCLC. Analysis of lung tissues from clinically-matched non-cancer patients is warranted. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5455. doi:1538-7445.AM2012-5455

Background: Approximately 50% of all non-small cell lung carcinoma (NSCLC) cases in the United States are adenocarcinoma histology. While definitive lung cancer resection offers a chance for cure in stage I and II NSCLC, up to 57% of patients will have disease relapse or metastatic disease within 5 years. A majority of the relapsed cases will take place within 2 years of definitive surgical resection. Thus, pathologic staging alone does not stratify patients for prognosis sufficiently. Identifying patients with aggressive versus indolent tumors would have critical implications for directing treatment and designing future clinical trials. We propose using microRNA (miRNA) for discovery and validation of prognostic biomarkers for stage I and II lung adenocarcinoma. Methods: For the Discovery Cohort, we identified treatment-naïve AJCC 7th edition stage I and II lung adenocarcinoma patient samples from 26 disease-free survivors (DFS) and overall survival (OS) at least 47 months and 14 patients with DFS and overall survival less than 23 months. Total RNA was extracted from formalin-fixed, paraffin-embedded (FFPE) samples. Differential miRNA array expression profiling between these two groups was assessed in the Discovery cohort on an 8x15 Agilent microarray printed with 886 miRNAs. Target miRNAs were selected based on minimum of 2-fold change in expression and Welch t-test p-value cut-off of 47 months and N=12 with DFS and OS47 months and N=4 DFS/OS Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4148. doi:1538-7445.AM2012-4148

Considered independently, lung cancer in never-smokers would rank among the ten most common causes of cancer mortality. Driver pathways and potential therapeutics must be identified for this clinically relevant subpopulation. We hypothesize that novel mutations and pathways identified by whole genome sequencing (WGS), whole transcriptome sequencing (WTS), and methylation profiling drive tumorigenesis in adenocarcinomas (AC) of never-smoker (NS) patients, and represent potential therapeutic targets. We have completed WGS, WTS, and methylation profiling on two lung ACs from female, never-smokers, one early-stage and one stage IV, and one female smoker (S) patient with early-stage lung AC. Approximately 100 short nucleotide variants (SNV) were discerned from the early-stage and stage IV NS lung ACs. Of interest, these NS patients lacked alterations in common genes associated with lung cancer such as EGFR and KRAS. In comparison, the lung AC from a smoker contained 78 SNVs, including a well-characterized KRAS mutation. Mutations in MAGEC1, a tumor marker in melanoma, were observed in common between the early-stage NS tumor and the smoker lung AC. The early-stage NS tumor contained a mutation in PIK3C3 and CSNK1E, a casein kinase involved Wnt signaling. The stage IV NS tumor demonstrated mutations in tumor suppressor genes such as p53, LATS2, and ATM. With relatively few mutations discerned from NS lung ACs, WTS and methylation profiling were performed. WTS showed 1,083 genes differentially regulated in the early-stage NS and Ingenuity Pathway Analysis implicated G-protein coupled receptor signaling. 2,000 genes were differentially expressed in the stage IV NS lung AC with sonic hedgehog implicated as a significantly regulated pathway. Besides gene expression, methylation profiling revealed ∼3,100 differentially methylated genes in the early-stage NS lung AC compared to normal lung DNA, with 65% hypo-methylated, including genes involved in lung carcinogenesis such as RET and BCL2. In the stage IV NS lung AC, ∼35,000 genes were differentially methylated compared to normal lung DNA (10X the early-stage NS) with ∼62% hypo-methylated. Thus, while an early-stage smoker lung AC displayed a classic driver mutation in KRAS, lung ACs from never-smokers had relatively quiet genomes. Despite few mutations, thousands of genes were differentially regulated at the mRNA level or by methylation. We continue to validate the genes implicated in NS lung AC by mutation, differential expression, and differential methylation, as well as integrating the data across platforms to discern significantly regulated pathways. We believe that the genes and pathways implicated by WGS, WTS, and methylation profiling will lead to a better understanding of NS lung AC and identify possible therapeutic interventions. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5068. doi:1538-7445.AM2012-5068

Background: Transitional cell carcinoma of the upper urinary tract (UUT) is a rare urothelial cancer, which most often affects males ages 50-70. Previous studies have shown some overlap with genetic alterations frequently observed in bladder cancer, however complete molecular characterization of UUT tumors is currently limited. A 20-year-old woman with treated acute lymphoblastic leukemia (ALL) in remission for ∼13 years, presented with present with numerous metastatic lesions involving bone, adrenal, retrocaval lymph node, and kidney. Biopsy confirmed a second primary carcinoma originating from the ureter or kidney. After two lines of systemic chemotherapy, the patient enrolled in a whole-genome (WGS) and whole transcriptome sequencing (WTS) pilot study. Methods: Tumor DNA and RNA was isolated from fresh-frozen tissue obtained by needle core biopsy of the right kidney tumor. Germline DNA was isolated from the patient's whole blood. Commercial benign ureter tissue was used for normal RNA comparison. Whole-genome paired-end library and transcriptome sequencing were done on Illumina's HiSeq 2000 platform. Burrows-Wheeler Transform algorithm was used in alignment to human reference genome (build 36). Several short nucleotide variants (SNVs) were selected for further validation with Sanger sequencing. Quantitative PCR with specific primers to either amplified or deleted genomic regions and ΔΔCt method were used to validate chromosomal copy number abnormalities. Results: WGS yielded 46X and 55X uniquely mappable reads for the tumor and normal samples, respectively. Seventy-one damaging somatic SNVs and a number of chromosomal aberrations were identified in regions of cancer relevant genes. Most notably, truncating mutations were found in p53 and KDM6A (oncogenic histone demethylase) genes as well as a damaging mutation in E2F8 transcription factor. KDM6A and E2F8 mutations were confirmed with bi-directional Sanger sequencing. A homozygous deletion encompassing the tumor suppressor p16 and a prominent focal 2MB high-level amplification at 7p11.2 that includes the EGFR oncogene were also detected. WTS produced ∼1006 reads for each sample. Expression analysis revealed loss-of-heterozygosity in p53 wild-type locus and a significant upregulation of EGFR. Additionally, a number of genes (MKI67, AURKA, TOP2A, BUB, FOXM, and PLK1) associated with cell cycle progression had >4-fold tumor overexpression. Genomic qPCR confirmed a 10-fold reduction in p16 while EGFR was amplified >40 times. Conclusion: Through WGS and WTS, a number of common oncogenic targets to be deregulated in this UUT, including p53 mutation, EGFR and MYC amplifications, and p16 deletion. These targets have previously been implicated in urothelial cancers, most notably in bladder. Given, negative KRAS mutation status, EGFR targeted therapy may be a reasonable treatment option for this patient. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3679. doi:1538-7445.AM2012-3679

Lung cancer remains the leading cause of cancer deaths in the US and throughout the world. Never-smokers with lung cancer constitute an understudied subset of these patients, though recent estimates show that ∼10% of men and women with lung cancer in the US are never-smokers. Independently, lung cancer in never-smokers ranks among the ten most common causes of cancer mortality, and thus the causes, driver pathways, and potential therapeutics must be investigated for this clinically relevant subpopulation. We hypothesize that novel mutations and pathways identified by whole genome sequencing (WGS) and whole transcriptome sequencing (WTS) drive tumorigenesis in adenocarcinomas of never-smoker patients, and represent potential therapeutic targets. We have completed WGS and WTS on two lung adenocarcinomas from female, never-smokers, one early-stage and one advanced-stage (metastatic), and one female smoker patient with early-stage lung adenocarcinoma. Approximately 100 short nucleotide variants (SNV) causing non-synonymous DNA sequence changes were discerned from the early- and advanced-stage adenocarcinomas from never-smokers. Of interest, these never-smoker patients lacked alterations in common genes associated with lung cancer such as EGFR, KRAS, and EML/ALK translocations, making them ideal cases for the discovery of new mutations that may drive lung adenocarcinomas in never-smokers. In comparison, the adenocarcinoma from a smoker patient contained 78 SNVs, including a well-characterized KRAS mutation. Mutations in MAGEC1, a tumor marker in melanoma and multiple myeloma, were observed in common between the early-stage, never-smoker tumor and the smoker tumor. The mutations observed in the never-smoker cases included genes suspected to play a role in lung carcinogenesis. The early-stage, never-smoker patient contained a mutation in PIK3C3 and DOCK10, known to play a role in cancer cell migration, and CSNK1E, a casein kinase involved Wnt signaling and in beta catenin-induced cancer cell proliferation. The late-stage never-smoker patient demonstrated a p53 mutation, a mutation in the tumor suppressor LATS2, and a mutation in the DNA damage checkpoint gene, ATM. The mutations discovered in known tumor suppressor genes tended to be in the late-stage, never-smoke patient. Ingenuity Pathway Analysis implicated G-protein coupled receptor signaling for the early-stage never-smoker, Hif1α signaling for the smoker, and Sonic Hedgehog Signaling for the late-stage, never-smoker in both WGS and WTS. Currently, we are validating the identified mutations discovered by WGS using Sanger sequencing and surveying their frequency in ∼30 and ∼60 additional cases of lung adenocarcinoma from never-smokers and clinically matched smokers, respectively. Validation of these mutations and pathways will lead to a better understanding of tumorigenesis and tumor progression in never-smokers and identify nodes for therapeutic vulnerability.

Background: Dysregulation of microRNAs (miRNAs) has been widely implicated in a variety of cancers and have been recognized as prognostic and/or predictive biomarkers. Lack of a standard method for stratifying advanced-stage non-small cell lung cancer (NSCLC) patients receiving platinum combination therapy often results in a number of patients that do not derive benefit yet are still exposed to treatment toxicity. We hypothesized that miRNAs in pre-treatment serum and/or plasma could be used to differentiate NSCLC patients who would have disease progression (PD) to first-line carboplatin and gemcitabine chemotherapy at first response assessment. Methods: miRNA array profiling containing probes in triplicate for 900 mature miRNAs (Sanger miRBASE v13.0 released in March 2009) and 450 precursor (pre-) miRNAs along with positive and negative control probes was performed on total RNA isolated from the pre-treatment serum and plasma of 24 advanced or unresectable NSCLC patients who went on to receive first-line carboplatin and gemcitabine chemotherapy. SAM data analysis was applied to find significantly differentially-expressed miRNAs in one condition (PD at first radiologic response) in contrast to another (those without disease progression [nonPD]). Data was normalized to U6 small nuclear RNA and the top differentially-expressed miRNAs were identified based on fold change, p-values, q-values, and false discovery rates. Differentially-expressed miRNAs were validated by quantitative PCR. Single validated candidates or combinations thereof were selected based on specificity and sensitivity to segregate patients with PD vs. nonPD. Correlations for clinical parameters with candidate miRNA were also assessed. Results: Four miRNAs were identified using miRNA microarray as potential candidate qualifiers. Two of them, pre-miR-518b and pre-miR-598, were validated in a qPCR assay and were shown to be significantly over-expressed in serum of PD patients. ROC curves plotting a combination of these two pre-miRNAs were able to discriminate between PD vs. nonPD patients with 83% sensitivity and 72% specificity. No significant correlation of these two pre-miRNAs was observed with clinical parameters such as age, gender, histology, smoking status, or overall survival. No significant plasma miRNA candidates were identified. Conclusion: Serum miRNAs may serve as a proof-of-concept screening tool in predicting chemoresistance to platinum-based combination chemotherapy. This is an important step towards personalized medicine and could enable stratification of patients to treatment options thus reducing exposure to specific toxic chemotherapy agents that in the end result would not benefit the patient. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 171. doi:10.1158/1538-7445.AM2011-171

Background One of the targets of a future multi-component vaccine against HIV-1 should be one of the virus' chief mucosal penetration processes, namely epithelial transcytosis. We further reported that fusion proteins based on the membrane proximal region (MPR) of gp41, playing a key role in the transcytotic process, and the mucosal targeting subunit B of cholera toxin (CTB) are successful immunogens eliciting such transcytosis blocking immune responses and that such proteins can be produced in plants