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Background Streptococcus pneumoniae interacts with numerous viral respiratory pathogens in the upper airway. It is unclear whether similar interactions occur with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Methods We collected saliva specimens from working-age adults undergoing SARS-CoV-2 molecular testing at outpatient clinics and via mobile community-outreach testing between July and November 2020 in Monterey County, California. After bacterial culture enrichment, we tested for pneumococci by means of quantitative polymerase chain reaction targeting the lytA and piaB genes, and we measured associations with SARS-CoV-2 infection using conditional logistic regression. Results Analyses included 1278 participants, with 564 enrolled in clinics and 714 enrolled through outreach-based testing. The prevalence of pneumococcal carriage was 9.2% (117 of 1278) among all participants (11.2% [63 of 564] in clinic-based testing and 7.6% [54 of 714] in outreach-based testing). The prevalence of SARS-CoV-2 infection was 27.4% (32 of 117) among pneumococcal carriers and 9.6% (112 of 1161) among noncarriers (adjusted odds ratio [aOR], 2.73 [95% confidence interval (CI): 1.58–4.69). Associations between SARS-CoV-2 infection and pneumococcal carriage were enhanced in the clinic-based sample (aOR, 4.01 [95% CI: 2.08–7.75]) and among symptomatic participants (3.38 [1.35–8.40]), compared with findings within the outreach-based sample and among asymptomatic participants. The adjusted odds of SARS-CoV-2 coinfection increased 1.24-fold (95% CI: 1.00–1.55-fold) for each 1-unit decrease in piaB quantitative polymerase chain reaction cycle threshold value among pneumococcal carriers. Finally, pneumococcal carriage modified the association of SARS-CoV-2 infection with recent exposure to a suspected coronavirus disease 2019 case (aOR, 7.64 [95% CI: 1.91–30.7] and 3.29 [1.94–5.59]) among pneumococcal carriers and noncarriers, respectively). Conclusions Associations of pneumococcal carriage detection and density with SARS-CoV-2 suggest a synergistic relationship in the upper airway. Longitudinal studies are needed to determine interaction mechanisms between pneumococci and SARS-CoV-2.

Purpose: Concomitant genetic alterations could account for transient clinical responses to tyrosine kinase inhibitors of the EGF receptor (EGFR) in patients harboring activating EGFR mutations.Experimental Design: We have evaluated the impact of pretreatment somatic EGFR T790M mutations, TP53 mutations, and Bcl-2 interacting mediator of cell death (BCL2L11, also known as BIM) mRNA expression in 95 patients with EGFR-mutant non–small-cell lung cancer (NSCLC) included in the EURTAC trial (trial registration: NCT00446225).Results: T790M mutations were detected in 65.26% of patients using our highly sensitive method based on laser microdissection and peptide-nucleic acid-clamping PCR, which can detect the mutation at an allelic dilution of 1 in 5,000. Progression-free survival (PFS) to erlotinib was 9.7 months for those with T790M mutations and 15.8 months for those without, whereas among patients receiving chemotherapy, it was 6 and 5.1 months, respectively (P < 0.0001). PFS to erlotinib was 12.9 months for those with high and 7.2 months for those with low/intermediate BCL2L11 expression levels, whereas among chemotherapy-treated patients, it was 5.8 and 5.5 months, respectively (P = 0.0003). Overall survival was 28.6 months for patients with high BCL2L11 expression and 22.1 months for those with low/intermediate BCL2L11 expression (P = 0.0364). Multivariate analyses showed that erlotinib was a marker of longer PFS (HR = 0.35; P = 0.0003), whereas high BCL2L11 expression was a marker of longer PFS (HR = 0.49; P = 0.0122) and overall survival (HR = 0.53; P = 0.0323).Conclusions: Low-level pretreatment T790M mutations can frequently be detected and can be used for customizing treatment with T790M-specific inhibitors. BCL2L11 mRNA expression is a biomarker of survival in EGFR-mutant NSCLC and can potentially be used for synthetic lethality therapies. Clin Cancer Res; 20(7); 2001–10. ©2014 AACR.

PDF file - 934K, Table S1. Quantification of T790M allele in our samples Table S2. Percentage of EGFR T790M mutation in each sample. Table S3. Results of the analysis of the T790M mutation in 705 paraffin-embedded lung tumor samples using our PNA method. Table S4. Primers for TP53 genotyping. Table S5. Cell lines used as controls for TP53 genotyping Table S6. Annealing conditions for TP53 genotyping. Table S7. Comparison of studies detecting the T790M mutation by different methods. Table S8. Summary of all systemic treatments after discontinuation of randomly assigned treatment in 95 patients included in the EURTAC study. Table S9. Response according to T790M status in both arms. Figure S1. Representation of deltaCt (mean) versus the ratio of the T790M allele in our in-house assay in (A, B) two separate dilution banks where the total amount of mutated DNA was 50 pg/microL and (C) in a dilution bank where the total amount of mutated DNA was 5 pg/microL. Figure S2. Progression-free survival according to T790M mutation status in (S1A) 50 patients treated with erlotinib and (S1B) 45 patients treated with chemotherapy. Figure S3. Progression-free survival according to T790M mutation status as determined by the TMDA in the entire cohort of 95 patients. Figure S4. Overall survival of 173 patients in the EURTAC trial at January 24, 2013. Figure S5. Overall survival of 83 patients with BIM expression data according to treatment arm and BIM mRNA expression levels.

BackgroundStreptococcus pneumoniae interacts with numerous viral respiratory pathogens in the upper airway. It is unclear whether similar interactions occur with SARS-CoV-2.MethodsWe collected saliva specimens from working-age adults receiving SARS-CoV-2 molecular testing at outpatient clinics and via mobile community-outreach testing between July and November 2020 in Monterey County, California. Following bacterial culture enrichment, we tested for pneumococci by quantitative polymerase chain reaction (qPCR) targeting the lytA and piaB genes, and measured associations with SARS-CoV-2 infection via conditional logistic regression.ResultsAnalyses included 1,278 participants, with 564 enrolled in clinics and 714 enrolled through outreach-based testing. Prevalence of pneumococcal carriage was 9.2% (117/1,278) among all participants (11.2% [63/564] clinic-based testing; 7.6% [54/714] outreach testing). Prevalence of SARS-CoV-2 infection was 27.4% (32/117) among pneumococcal carriers and 9.6% (112/1,161) among non-carriers (adjusted odds ratio [aOR]: 2.73; 95% confidence interval: 1.58-4.69). Associations between SARS-CoV-2 infection and pneumococcal carriage were enhanced in the clinic-based sample (aOR=4.01 [2.08-7.75]) and among symptomatic participants (aOR=3.38 [1.35-8.40]), when compared to findings within the outreach-based sample and among asymptomatic participants. Adjusted odds of SARS-CoV-2 co-infection increased 1.24 (1.00-1.55)-fold for each 1-unit decrease in piaB qPCR CT value among pneumococcal carriers. Last, pneumococcal carriage modified the association of SARS-CoV-2 infection with recent exposure to a suspected COVID-19 case (aOR=7.64 [1.91-30.7] and 3.29 [1.94-5.59]) among pneumococcal carriers and non-carriers, respectively).ConclusionsAssociations of pneumococcal carriage detection and density with SARS-CoV-2 suggest a synergistic relationship in the upper airway. Longitudinal studies are needed to determine interaction mechanisms between pneumococci and SARS-CoV-2.Key pointsIn an adult ambulatory and community sample, SARS-CoV-2 infection was more prevalent among pneumococcal carriers than non-carriers.Associations between pneumococcal carriage and SARS-CoV-2 infection were strongest among adults reporting acute symptoms and receiving SARS-CoV-2 testing in a clinical setting.

For the past 40 years, progress in rhabdomyosarcoma (RMS) has been focused on understanding its molecular basis and characterizing the mutations that drive its tumorigenesis and progression. Genetic predisposition to RMS has allowed discovery of key genetic pathways and driver mutations. Subclassification of RMS into embryonal (ERMS) and alveolar (ARMS) subtypes has shifted from histology to PAX-FOXO1 fusion status, and new driver mutations have been found in spindle cell RMS. Comprehensive molecular profiling leveraging genome-scale next-generation sequencing (NGS) indicates that the RAS/RAF/PI3K axis is mutated in the majority of ERMS and modulated by downstream effects of PAX-FOXO1 fusions in ARMS. Because of the continued poor outcome of high-risk RMS, a variety of molecular targets have been or are now being tested in current or recent therapy trials. New techniques such as single cell sequencing, spatial multi-omics, and CRISPR/Cas9 genome editing offer potential for further discovery, but a need for clinically annotated specimens persists.

SummaryRegular surveillance testing of asymptomatic individuals for SARS-CoV-2 has played a vital role in SARS-CoV-2 outbreak prevention on college and university campuses. Here we describe the voluntary saliva testing program instituted at the University of California, Berkeley during an early period of the SARS-CoV-2 pandemic in 2020. The program was administered as a research study ahead of clinical implementation, enabling us to launch surveillance testing while continuing to optimize the assay. Results of both the testing protocol itself and the study participants’ experience show how the program succeeded in providing routine, robust testing capable of contributing to outbreak prevention within a campus community and offer strategies for encouraging participation and a sense of civic responsibility.

Rhabdomyosarcoma, the most common soft tissue sarcoma in childhood, has challenged and intrigued soft tissue pathologists ever since the original descriptions. Once based on the identification of rhabdomyoblastic cells with elongate eosinophilic cytoplasm, the diagnosis has evolved to include tumors composed only of primitive mesenchymal cells but now relies heavily on immunohistochemical stains for desmin, myogenin, and MyoD. Rhabdomyosarcomas show a variety of histological patterns, giving rise to classifications that have included embryonal, alveolar, botryoid, pleomorphic, spindle cell, and sclerosing subtypes. These have been linked to prognosis and treatment assignment in the past, but that concept has been superseded by the identification of PAX3-FOXO1 or PAX7-FOXO1 fusions. Fusion testing results are more predictive of outcome and have become standard practice in clinical management. However, high risk tumors with alveolar histology or metastatic disease continue to resist oncologic treatment.

Commonly used RT-qPCR-based SARS-CoV-2 diagnostics require 2-3 separate reactions or rely on detection of a single viral target, adding time and cost or risk of false-negative results. Currently, no test combines detection of widely used SARS-CoV-2 E- and N-gene targets and a sample control in a single, multiplexed reaction. We developed the IGI-LuNER RT-qPCR assay using the Luna Probe Universal One-Step RT-qPCR master mix with publicly available primers and probes to detect SARS-CoV-2 N gene, E gene, and human RNase P (NER). This combined, cost-effective test can be performed in 384-well plates with detection sensitivity suitable for clinical reporting, and will aid in future sample pooling efforts, thus improving throughput of SARS-CoV-2 detection.Graphical Abstract

Pediatric sarcomas constitute one of the largest groups of childhood cancers, following hematopoietic, neural, and renal lesions. Partly because of their diversity, they continue to offer challenges in diagnosis and treatment. In spite of the diagnostic, nosologic, and therapeutic gains made with genetic technology, newer means for investigation are needed. This article reviews emerging technology being used to study human neoplasia and how these methods might be applicable to pediatric sarcomas. Methods reviewed include single cell RNA sequencing (scRNAseq), spatial multi-omics, high-throughput functional genomics, and clustered regularly interspersed short palindromic sequence-Cas9 (CRISPR-Cas9) technology. In spite of these advances, the field continues to be challenged by a dearth of properly annotated materials, particularly from recurrences and metastases and pre- and post-treatment samples.

A widespread approach to modern cancer therapy is to identify a single oncogenic driver gene and target its mutant-protein product (for example, EGFR-inhibitor treatment in EGFR-mutant lung cancers). However, genetically driven resistance to targeted therapy limits patient survival. Through genomic analysis of 1,122 EGFR-mutant lung cancer cell-free DNA samples and whole-exome analysis of seven longitudinally collected tumor samples from a patient with EGFR-mutant lung cancer, we identified critical co-occurring oncogenic events present in most advanced-stage EGFR-mutant lung cancers. We defined new pathways limiting EGFR-inhibitor response, including WNT/β-catenin alterations and cell-cycle-gene (CDK4 and CDK6) mutations. Tumor genomic complexity increases with EGFR-inhibitor treatment, and co-occurring alterations in CTNNB1 and PIK3CA exhibit nonredundant functions that cooperatively promote tumor metastasis or limit EGFR-inhibitor response. This study calls for revisiting the prevailing single-gene driver-oncogene view and links clinical outcomes to co-occurring genetic alterations in patients with advanced-stage EGFR-mutant lung cancer.

Activation of the phosphoinositide 3-kinase (PI3K) pathway occurs widely in human cancers. Although somatic mutations in the PI3K pathway genes PIK3CA and PTEN are known to drive PI3K pathway activation and cancer growth, the significance of somatic mutations in other PI3K pathway genes is less clear. Here, we establish the signaling and oncogenic properties of a recurrent somatic mutation in the PI3K p110β isoform that resides within its kinase domain (PIK3Cβ(D1067V)). We initially observed PIK3Cβ(D1067V) by exome sequencing analysis of an EGFR-mutant non-small cell lung cancer (NSCLC) tumor biopsy from a patient with acquired erlotinib resistance. On the basis of this finding, we hypothesized that PIK3Cβ(D1067V) might function as a novel tumor-promoting genetic alteration, and potentially an oncogene, in certain cancers. Consistent with this hypothesis, analysis of additional tumor exome data sets revealed the presence of PIK3Cβ(D1067V) at low frequency in other patient tumor samples (including renal cell carcinoma, glioblastoma multiforme, head and neck squamous cell carcinoma, melanoma, thyroid carcinoma and endometrial carcinoma). Functional studies revealed that PIK3Cβ(D1067V) promoted PI3K pathway signaling, enhanced cell growth in vitro, and was sufficient for tumor formation in vivo. Pharmacologic inhibition of PIK3Cβ with TGX-221 (isoform-selective p110β inhibitor) specifically suppressed growth in patient-derived renal-cell carcinoma cells with endogenous PIK3Cβ(D1067V) and in NIH-3T3 and human EGFR-mutant lung adenocarcinoma cells engineered to express this mutant PI3K. In the EGFR-mutant lung adenocarcinoma cells, expression of PIK3Cβ(D1067V) also promoted erlotinib resistance. Our data establish a novel oncogenic form of PI3K, revealing the signaling and oncogenic properties of PIK3Cβ(D1067V) and its potential therapeutic relevance in cancer. Our findings provide new insight into the genetic mechanisms underlying PI3K pathway activation in human tumors and indicate that PIK3Cβ(D1067V) is a rational therapeutic target in certain cancers.

The current understanding of tumorigenesis is largely centered on a monogenic driver oncogene model. This paradigm is incompatible with the prevailing clinical experience in most solid malignancies: monotherapy with a drug directed against an individual oncogenic driver typically results in incomplete clinical responses and eventual tumor progression1-7. By profiling the somatic genetic alterations present in over 2,000 cases of lung cancer, the leading cause of cancer mortality worldwide8,9, we show that combinations of functional genetic alterations, i.e. genetic collectives dominate the landscape of advanced-stage disease. We highlight this polygenic landscape and evolution of advanced-stage non-small cell lung cancer (NSCLC) through the spatial-temporal genomic profiling of 7 distinct tumor biopsy specimens and 6 plasma specimens obtained from an EGFR-mutant NSCLC patient at (1) initial diagnosis of early-stage disease, (2) metastatic progression, (3) sequential treatment and resistance to 2 EGFR inhibitors, (4) death. The comprehensive genomic analysis of this case, coupled with circulating free (cf) tumor DNA profiling of additional advanced-stage EGFR-mutant NSCLC clinical cohorts with associated treatment responses uncovered features of evolutionary selection for multiple concurrent gene alterations: including the presence of EGFR inhibitor-sensitive (EGFRL858R;EGFRexon19del) or inhibitor-resistant (EGFRT790M;EGFRC797S) forms of oncogenic EGFR along with cell cycle gene alterations (e.g. in CDK4/6, CCNE1, RB1) and activating alterations in WNT/β-catenin and PI3K pathway genes, which our data suggest can cooperatively impart non-redundant functions to limit EGFR targeted therapy response and/or promote tumor progression. Moreover, evidence of an unanticipated parallel evolution of both EGFRT790M and two distinct forms of oncogenic PIK3CA was observed. Our study provides a large-scale clinical and genetic dataset of advanced-stage EGFR-mutant NSCLC, a rationale for specific polytherapy strategies such as EGFR and CDK4/6 inhibitor co-treatment to potentially enhance clinical outcomes, and prompts a re-evaluation of the prevailing paradigm of monogenic-based molecular stratification for targeted therapy. Instead, our findings highlight an alternative model of genetic collectives that operate through epistasis to drive lung cancer progression and therapy resistance.

Here we report that the transcription factor cyclic AMP-responsive element-binding protein H (CREB-H, encoded by CREB3L3) is required for the maintenance of normal plasma triglyceride concentrations. CREB-H-deficient mice showed hypertriglyceridemia secondary to inefficient triglyceride clearance catalyzed by lipoprotein lipase (Lpl), partly due to defective expression of the Lpl coactivators Apoc2, Apoa4 and Apoa5 (encoding apolipoproteins C2, A4 and A5, respectively) and concurrent augmentation of the Lpl inhibitor Apoc3. We identified multiple nonsynonymous mutations in CREB3L3 that produced hypomorphic or nonfunctional CREB-H protein in humans with extreme hypertriglyceridemia, implying a crucial role for CREB-H in human triglyceride metabolism.

e18589Background: Despite the critical need for cancer patients to participate in clinical trials, only 3% of US cancer patients actually do so, and 40-60% of oncology clinical trials fail to recru...

Nhp6A is an abundant non-histone chromatin-associated protein in Saccharomyces cerevisiae that contains a minor groove DNA binding motif called the HMG box. In this report, we show that Nhp6Ap binds to cisplatin intrastrand cross-links on duplex DNA with a 40-fold greater affinity than to unmodified DNA with the same sequence. Nevertheless, Nhp6Ap bound to cisplatinated DNA readily exchanges onto unmodified DNA. Phenanthroline-copper footprinting and two-dimensional NMR on complexes of wild-type and mutant Nhp6Ap with DNA were employed to probe the mode of binding to the cisplatin lesion. Recognition of the cisplatin adduct requires a surface-exposed phenylalanine on Nhp6Ap that promotes bending of DNA by inserting into the helix from the minor groove. We propose that Nhp6Ap targets the cisplatin adduct by means of intercalation by the phenylalanine and that it can bind in either orientation with respect to the DNA lesion. A methionine, which also inserts between base pairs and functions in target selection on unmodified DNA, plays no apparent role in recognition of the cisplatin lesion. Basic amino acids within the N-terminal arm of Nhp6Ap are required for high-affinity binding to the cisplatin adduct as well as to unmodified DNA. Cisplatin mediates its cytotoxicity by forming covalent adducts on DNA, and we find that Deltanhp6a/b mutants are hypersensitive to cisplatin in comparison with the wild-type strain. In contrast, Deltanhp6a/b mutants are slightly more resistant to hydrogen peroxide and ultraviolet irradiation. Therefore, Nhp6A/Bp appears to directly or indirectly function in yeast to enhance cellular resistance to cisplatin.

Purpose: Concomitant genetic alterations could account for transient clinical responses to tyrosine kinase inhibitors of the EGF receptor (EGFR) in patients harboring activating EGFR mutations. Experimental Design: We have evaluated the impact of pretreatment somatic EGFR T790M mutations, TP53 mutations, and Bcl-2 interacting mediator of cell death (BCL2L11, also known as BIM) mRNA expression in 95 patients with EGFR-mutant non–small-cell lung cancer (NSCLC) included in the EURTAC trial (trial registration: NCT00446225). Results: T790M mutations were detected in 65.26% of patients using our highly sensitive method based on laser microdissection and peptide-nucleic acid-clamping PCR, which can detect the mutation at an allelic dilution of 1 in 5,000. Progression-free survival (PFS) to erlotinib was 9.7 months for those with T790M mutations and 15.8 months for those without, whereas among patients receiving chemotherapy, it was 6 and 5.1 months, respectively (P < 0.0001). PFS to erlotinib was 12.9 months for those with high and 7.2 months for those with low/intermediate BCL2L11 expression levels, whereas among chemotherapy-treated patients, it was 5.8 and 5.5 months, respectively (P = 0.0003). Overall survival was 28.6 months for patients with high BCL2L11 expression and 22.1 months for those with low/intermediate BCL2L11 expression (P = 0.0364). Multivariate analyses showed that erlotinib was a marker of longer PFS (HR = 0.35; P = 0.0003), whereas high BCL2L11 expression was a marker of longer PFS (HR = 0.49; P = 0.0122) and overall survival (HR = 0.53; P = 0.0323). Conclusions: Low-level pretreatment T790M mutations can frequently be detected and can be used for customizing treatment with T790M-specific inhibitors. BCL2L11 mRNA expression is a biomarker of survival in EGFR-mutant NSCLC and can potentially be used for synthetic lethality therapies. Clin Cancer Res; 20(7); 2001–10. ©2014 AACR.

Nucleic acid extraction is a key step in many molecular laboratories. Obtaining DNA and RNA of sufficient quality and quantity is critical for the next-generation sequencing assays performed at our facility. To improve efficiency of our lab process, we implemented automated nucleic acid …

Industries strewn across all fields are constantly striving to improve on processes, reduce waste, and increase efficiency. During this pursuit, two common challenges almost always present themselves to companies both big and small: 1) the goal of introducing automation efficiently, and 2) the goal of eliminating or reducing paper. The potential benefits brought on by accomplishing these …

Would a bricklayer build a better house if he met the family who would inhabit it? Would drug developers create drugs faster if they spoke to the patients whom it would soon be prescribed to? How would job satisfaction improve in a restaurant if compliments went to the entire staff and not just the chef? We wanted to answer …

Developing a more robust approach to measure mutational burden is of central importance to improving the characterization of the molecular profile of tumor and may improve our ability to predict tumor progression or response to therapy in patients. Mutational burden is typically calculated as a direct enumeration of called somatic mutations per megabase covered. However, there is a growing appreciation that tumor purity, variable sequencing coverage, and copy number alterations can substantially impact the accurate identification any specific somatic mutation. Furthermore, population genetic theory and empirical data indicate that in many cases the vast majority of somatic mutations appear in only a small subpopulation of tumor cells, a context in which there is a high likelihood that an individual subclonal mutation may not be identified by conventional analysis. This tendency to miss low frequency mutations is highly variable and dependent, in part, upon sample purity and results in a strong source of bias not addressed in existing methods to measure variant allele frequencies. We present a novel computational method that incorporates these sources of bias in a coherent probabilistic framework that enables maximum-likelihood inference of relevant population parameters such as mutation burden. We apply our method to simulated data as well as patient tumor samples diluted with varying known proportions of normal DNA. We show that our approach allows us to generate estimates of mutation burden that are robust to the substantial variations in purity and sequencing coverage that are frequently encountered in patient tumor analysis. Hence, our novel method may improve the accurate detection and quantification of variant alleles in patient tumors to better understand their genetic landscape and guide clinical management. Citation Format: Oscar Westesson, Rasmus Nielsen, John St John, Aleah Caulin, Nicholas Hahner, Stewart Stewart, Catherine Foo, Kimberly Lung, Jeff Catalano, Mandy Lee, Petros Giannikopoulos, Will Polkinghorn, Jonathan Wiessman, Aviv Regev, Trever Bivona. Robust estimation of mutation burden. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2173. doi:10.1158/1538-7445.AM2015-2173

The retroesophageal right subclavian artery is an anatomical abnormality encountered by anatomists and pathologists and recently interventional cardiologists and thoracic surgeons have also come across this phenomenon. We report a case of a retroesophageal right subclavian artery arising from a normally located left aortic arch in a young male autopsied in the Department of Forensic Service of Warsaw Medical University. In addition to the aforementioned anomaly, the presence of a right non-recurrent inferior laryngeal nerve was noticed. The possible embryonic development of these branching patterns and their clinical significance is discussed.

Noonan syndrome is a rare disease, mainly presenting with malformations such as dysplasia and stenosis of the pulmonary valve, atrial septal defect and a typical pattern of hypertrophic cardiomyopathy. We report a case of Noonan syndrome with giant coronary aneurysms.A young woman with the phenotypic characteristics of Noonan's syndrome presented with severe pulmonary stenosis and giant coronary aneurysms. Cross sectional echocardiography showed valvar and subvalvar pulmonary stenosis. The valve itself was thickened and dysplastic, a characteristic that is typical of Noonan's syndrome. In addition to the usual abnormalities of the pulmonary valve and the ventricular myocardium, the patient showed a wide spectrum of previously unreported coronary aneurysms.These additional findings support the hypothesis that a vasculitic process has been superimposed on the connective tissue defect associated with Noonan's syndrome. Furthermore, since the pathogenesis of the condition remains unclear, this case stresses the need to look carefully for abnormalities co-expressed in Noonan's syndrome.

Background: NSCLC p with EGFR mutations initially respond to EGFR tyrosine kinase inhibitors (TKIs) but ultimately relapse. Sub-genomic molecular studies indicate that the EGFR T790M mutation and the activation of MET, PI3K, AXL, HER2 and MAPK can lead to acquired resistance to EGFR TKIs. To date, no integrated comprehensive genomic investigation of EGFR TKI resistance has been reported. Methods: FFPE biopsies of erlotinib-sensitive and erlotinib-resistant tumors were obtained from 16 EGFR mutant NSCLC patients. The samples were analyzed by whole exome sequencing and whole transcriptome sequencing utilizing the Illumina HiSeq2500 platform. In addition, targeted gene sequencing was performed with the Illumina TruSeq Amplicon-Cancer Panel and run on the MiSeq system. Results: Erlotinib resistant NSCLC specimens harbored known resistance drivers, including EGFR T790M mutations (6/16; 38%), MET amplification (2/16; 13%), and AXL upregulation (3/16; 19%). Differential expression analysis between resistant and pre-treatment states revealed enrichment in the post-resistant tumors of ERBB2, and FGFR signaling pathways. Biopsies from patients that developed an EGFR T790M mutation post resistance exhibited enrichment in pathways associated with cell cycle, meiosis, telomere maintenance and transcriptional regulation. Copy number analysis demonstrated amplifications in the post-resistance setting that correlated significantly with progression free survival, including regions containing NFKBIA, NKX2-1, and TSC-1. There was also strong correlation between the copy number changes observed and the expression mRNA levels of specific genes. Of note, each resistant tumor exhibited greater copy number similarity to the corresponding matched pre-treatment sample compared to other tumors within the resistance cohort. Conclusions: We conducted the first ever comprehensive integrated genomic analysis of EGFR TKI resistant NSCLC patients, and identified both known and potentially novel drivers of EGFR TKI resistance. This study demonstrated the feasibility and utility of comprehensive genomic analysis in the clinical management of NSCLC receiving targeted therapy. Together, our data provide unprecedented insight into the molecular pathogenesis of escape from EGFR oncogene inhibition in NSCLC. We are now conducting a prospective observational study in additional NSCLC patients on targeted therapy. Citation Format: Petros Giannikopoulos, John St. John, Nicholas Hahner, Joel S. Parker, Niki Karachaliou, Carlota Costa, Oscar Westesson, Urvish Parikh, Catherine K. Foo, Aleah F. Cauhlin, Maria D. Lozano, Santiago Viteri, Jose L. Perez-Gracia, Alessandra Curioni, Eloisa Jantus-Lewintre, Carlos Camps, Alain Vergenegre, Radj Gervais, Anne Wellde, Jonathan Barry, George W. Wellde, Rodolfo Bordoni, Rolf Stahel, Andres Felipe Cardona Zorilla, William R. Polkinghorn, Jonathan Weissman, Trever G. Bivona, Rafael Rosell. Integrated genomic analysis by whole exome and transcriptome sequencing of tumor samples from EGFR-mutant non-small-cell lung cancer patients with acquired resistance to erlotinib. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 954. doi:10.1158/1538-7445.AM2014-954

Despite recent advances in the understanding of the biology and genetics of lung cancer, and despite the introduction of multiplex somatic mutation testing in the clinic, the long-term survival for all lung cancer patients, particularly for those with advanced disease, remains low. Lung cancer is the leading cause of cancer death globally, resulting in 1.4 million deaths annually, including 165,000 patients in the United States per year. In order to address the critical need for comprehensive profiling of these patients, we developed a novel, CLIA-certified, whole exome and low-coverage whole genome sequencing assay that applies a disease-focused, integrated approach to identify therapeutically actionable drivers of disease. A panel (12) of surgically resected NSCLC specimens along with corresponding adjacent normal tissue underwent DNA extraction in a clinical (CLIA) environment. Tumor and normal genomic DNA was prepared for whole exome sequencing using the using the Agilent SureSelectXT Human All Exon V5 kit according to the manufacturer's instructions, and libraries were sequenced on the Illumina HiSeq2500 at an average depth of 500X. Genomic DNA was then prepared for whole genome sequencing using Illumina's Nextera system and run on the Illumina HiSeq2500 platform at an average depth of 1-2X. Somatic variants were detected using Strelka and somatic copy number alterations (SCNAs) were identified using a novel algorithm comparing normalized read counts within genomic segments as well as genes in the tumor to a panel of normal tissues. In parallel, the same tumor/normal specimens were analyzed by two separate CLIA laboratories via 1) a clinically validated Ion Torrent AmpliSeq Cancer Panel assay, and 2) a clinically validated cancer-focused, high-resolution comparative genome hybridization (CGH) array. In addition, a well-characterized panel of 10 germline samples obtained from the 1000 Genomes Project were pooled to simulate a broad spectrum of somatic single nucleotide variant and indel allele frequencies. Sequencing, data analysis and clinical reporting were completed for all 12 cases with an average turnaround time of less than 3 weeks. Single nucleotide variants and indels were identified with an accuracy of greater than 99%, with a limit of detection of 5-10% mutant allele frequency. Somatic copy number alterations were observed with an overall accuracy of greater than 95%. Actionable variants were identified by cross-referencing individual results with our internally developed, lung-cancer focused therapeutic association database. Citation Format: Catherine K. Foo, John St. John, Nicholas Hahner, Oscar Westesson, Mitchell E. Skinner, Urvish Parikh, Kimberly Lung, Aleah F. Cauhlin, Jeffrey P. Catalano, Anne S. Wellde, Jonathan K. Barry, George W. Wellde, Patrick Ma, Rafael Rosell, Andres Felipe Cardona Zorilla, William R. Polkinghorn, Trever G. Bivona, Jonathan S. Weissman, Petros Giannikopoulos. Comprehensive integrated genomic analysis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4707. doi:10.1158/1538-7445.AM2014-4707

8083 Background: Patients with EGFR-mutant NSCLC often respond initially to EGFR kinase inhibitor therapy but ultimately relapse. The spectrum of clinical resistance mechanisms and mechanistically ...

e22122 Background: Despite recent advances in the understanding of the biology and genetics of lung cancer, and despite the introduction of multiplex somatic mutation testing in the clinic, the lon...

11010 Background: NSCLC p with EGFR mutations initially respond to EGFR tyrosine kinase inhibitors (TKIs) but ultimately relapse. Sub-genomic molecular studies indicate that the EGFR T790M mutation and the activation of MET, PI3K, AXL, HER2 and MAPK can lead to acquired resistance to EGFR TKIs. To date, no integrated comprehensive genomic investigation of EGFR TKI resistance has been performed. Methods: FFPE biopsies of erlotinib-sensitive and erlotinib-resistant tumors were obtained from 11 EGFR mutant NSCLC p. DNA was extracted from all tumor and corresponding normal tissue samples and underwent whole exome sequencing using the Illumina HiSeq2500. RNA was extracted from all tumor samples and analyzed by whole transcriptome sequencing, also using the Illumina HiSeq2500. Results: Erlotinib resistant NSCLC specimens harbored upregulation of known resistance drivers including MET and AXL and novel alterations including upregulation of genes that are: 1) recurrently mutated in NSCLC, including ALK, STK11; 2) components of established embryonic stem cell signatures, including targets of Nanog, Oct4, Sox2, c-Myc, 3) neuronal lineage specific regulators, including NTRK3, NRCAM, ALK, LRP4. The analysis also revealed downregulation of several genes that are: 1) components of innate and acquired immunity, including HLA-A, -B, DQ, CD40; 2) phosphatases regulating survival signaling pathways, including PTEN, PTPRD, 3) proapoptotic components, including BNIP3L, IKIP. Conclusions: This study demonstrated the feasibility and utility of comprehensive genomic analysis in the clinical management of NSCLC p receiving targeted therapy. We identified known and novel molecular biomarkers of erlotinib acquired resistance in NSCLC p, and uncovered a previously unappreciated role for genetic events governing stem cell and neuronal phenotypes as well as immune evasion in erlotinib acquired resistance in NSCLC p. Together, our data provide unprecedented insight into the molecular pathogenesis of escape from EGFR oncogene inhibition in NSCLC. We are now conducting a prospective observational study in additional NSCLC p.

11067 Background: Major obstacles limiting the clinical success of EGFR TKI therapy in non-small cell lung cancer (NSCLC) patients are heterogeneity and variability in the initial anti-tumor response to treatment. The underlying molecular basis for this heterogeneity has not been explored in patients immediately after initiation of therapy. Methods: We conducted CT-guided core needle biopsies immediately prior to erlotinib treatment initiation and at 6 days post erlotinib initiation in a patient with histologically confirmed NSCLC harboring an established activating mutation in EGFR. DNA from the paired frozen biopsies and matched normal tissue was analyzed by whole exome sequencing and RNA from the biopsies was analyzed by whole transcriptome sequencing. High-resolution CT images were obtained at the time of each biopsy to compare the degree of molecular and radiographic response observed. Results: Two established activating somatic mutations were identified in EGFR (p.G719A and p. R776H). Selective depletion of each EGFR mutant allele, but not the EGFR wild type allele, was observed upon erlotinib treatment. Gene expression analysis of the paired transcriptomes revealed that erlotinib treatment resulted in significant upregulation of proapoptotic genes including BAD, BAX, BID, CASP3 and growth inhibitory genes including CDKN1A, GADD45B, GADD45G and downregulation of growth-promoting genes including CCNB1 and CCND3. Several unexpected and novel molecular biomarkers were identified by transcriptome analysis and the complete dataset will be presented. High-resolution CT scans revealed no interval radiographic changes in the target lesion and no clinical complications were encountered. Conclusions: This study is the first reported integrated genomic analysis of EGFR-mutant NSCLC immediately following EGFR TKI initiation. We documented the feasibility, safety and utility of this strategy to establish initial drug efficacy at the molecular level prior to any radiographic evidence of response. Additional, serial integrated genomic analysis is ongoing in the index patient and others on therapy to enhance the management of NSCLC patients on targeted therapy.

Pleuropulmonary blastoma (PPB) is a rare, aggressive childhood cancer arising from lung mesenchymal cells. In early stages, these cells reside in the walls of epithelial-lined cysts and may remain dormant or progress to thickened cysts and then highly malignant masses over several years. Familial germline loss-of-function DICER1 variants have been described, although carriers may not be affected. Therefore, we sought to uncover additional genetic events underlying tumor development through whole exome sequencing of 15 tumor/normal pairs. In addition to known loss-of-function germline variants found throughout DICER1, this analysis identified somatic missense mutations clustered in the DICER1 RNase IIIb domain in nearly every case. While the somatic mutations impact novel amino acid residues, expression analysis demonstrated depletion of 5p-derived miRNAs similar to that described in other DICER1-mutant tumors. While mutation rates were relatively high compared to other pediatric solid tumors, most mutations, except those in DICER1 and TP53, appeared to be subclonal. Early stage tumors (PPB Type II) had predominantly single-copy deletion of TP53 whereas late stage tumors (PPB Type III) had secondary TP53 mutations. Several late stage tumors also had activating mutations in RAS pathway members, including NRAS and BRAF. Therefore, compound DICER1 loss appears to be initiating and synergistic with incremental loss of TP53 and acquisition of additional driver mutations. We subsequently characterized this molecular pattern using deep sequencing and immunohistochemistry in an extension cohort of 45 additional tumors. This step-wise series of germline and somatic alterations illustrates a genetic mechanism consistent with the progressive nature of pleuropulmonary blastoma. Citation Format: Trevor J. Pugh, Weiying Yu, Jiandong Yang, Lauren Ambrogio, Scott L. Carter, Adam Kiezun, Aaron McKenna, Petros Giannikopoulos, Gad Getz, Yoav Messinger, Matthew Meyerson, D. Ashley Hill. Progressive biallelic loss of TP53 is associated with progression of pleuropulmonary blastoma initiated by germline loss and somatic mutation of DICER1. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3806. doi:10.1158/1538-7445.AM2013-3806