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Supplementary Tables 1-3 Supplementary Table 1. Location and number of NSCLC, normal and field of cancerization specimens analyzed for genome-wide allelic imbalance Supplementary Table 2. Summary of identified allelic imbalance events in the normal-appearing airway field of cancerization in early-stage NSCLC Supplementary Table 3. Airway events with negative B-allele frequency correlations

Supplementary information including supplementary methods, supplementary tables and figures as well as supplementary figure legends. Supplementary Figure 1. HapLOH results for matched normal large airway and NSCLC tumor from case 18 Supplementary Figure 2. Distribution of posterior probabilities for airway events called by hapLOH Supplementary Figure 3: Allelic imbalance events detected in normal-appearing airway brushings Supplementary Figure 4. Event classification using B-allele frequencies and log R ratios Supplementary Figure 5. Effect of allelic imbalance on B-allele frequencies Supplementary Figure 6: Distribution of focal and arm airway events

Visually normal cells adjacent to, and extending from, tumors of the lung may carry molecular alterations characteristics of the tumor itself, an effect referred to as airway field of cancerization. This airway field has been postulated as a model for early events in lung cancer pathogenesis. Yet the genomic landscape of somatically acquired molecular alterations in airway epithelia of lung cancer patients has remained unknown. To begin to fill this void, we sought to comprehensively characterize the genomic architecture of chromosomal alterations inducing allelic imbalance (AI) in the airway field of the most common type of lung tumors, non–small cell lung cancer (NSCLC). To do so, we conducted a genome-wide survey of multiple spatially distributed normal-appearing airways, multiregion tumor specimens, and uninvolved normal tissues or blood from 45 patients with early-stage NSCLC. We detected alterations in airway epithelia from 22 patients, with an increased frequency in NSCLCs of squamous histology. Our data also indicated a spatial gradient of AI in samples at closer proximity to the NSCLC. Chromosome 9 displayed the highest levels of AI and comprised recurrent independent events. Furthermore, the airway field AI included oncogenic gains and tumor suppressor losses in known NSCLC drivers. Our results demonstrate that genome-wide AI is common in the airway field of cancerization, providing insights into early events in the pathogenesis of NSCLC that may comprise targets for early treatment and chemoprevention. Cancer Res; 76(13); 3676–83. ©2016 AACR.

Background Triple-negative breast cancer (TNBC) is a heterogeneous disease and we have previously shown that rapid relapse of TNBC is associated with distinct sociodemographic features. We hypothesized that rapid versus late relapse in TNBC is also defined by distinct clinical and genomic features of primary tumors. Methods Using three publicly-available datasets, we identified 453 patients diagnosed with primary TNBC with adequate follow-up to be characterized as ‘rapid relapse’ (rrTNBC; distant relapse or death ≤2 years of diagnosis), ‘late relapse’ (lrTNBC; > 2 years) or ‘no relapse’ (nrTNBC: > 5 years no relapse/death). We explored basic clinical and primary tumor multi-omic data, including whole transcriptome (n = 453), and whole genome copy number and mutation data for 171 cancer-related genes (n = 317). Association of rapid relapse with clinical and genomic features were assessed using Pearson chi-squared tests, t-tests, ANOVA, and Fisher exact tests. We evaluated logistic regression models of clinical features with subtype versus two models that integrated significant genomic features. Results Relative to nrTNBC, both rrTNBC and lrTNBC had significantly lower immune signatures and immune signatures were highly correlated to anti-tumor CD8 T-cell, M1 macrophage, and gamma-delta T-cell CIBERSORT inferred immune subsets. Intriguingly, lrTNBCs were enriched for luminal signatures. There was no difference in tumor mutation burden or percent genome altered across groups. Logistic regression mModels that incorporate genomic features significantly outperformed standard clinical/subtype models in training (n = 63 patients), testing (n = 63) and independent validation (n = 34) cohorts, although performance of all models were overall modest. Conclusions We identify clinical and genomic features associated with rapid relapse TNBC for further study of this aggressive TNBC subset.

BackgroundCirculating tumor DNA (ctDNA) offers minimally invasive means to repeatedly interrogate tumor genomes, providing opportunities to monitor clonal dynamics induced by metastasis and therapeutic selective pressures. In metastatic cancers, ctDNA profiling allows for simultaneous analysis of both local and distant sites of recurrence. Despite the promise of ctDNA sampling, its utility in real-time genetic monitoring remains largely unexplored.MethodsIn this exploratory analysis, we characterize high-frequency ctDNA sample series collected over narrow time frames from seven patients with metastatic triple-negative breast cancer, each undergoing treatment with Cabozantinib, a multi-tyrosine kinase inhibitor (NCT01738438,https://clinicaltrials.gov/ct2/show/NCT01738438). Applying orthogonal whole exome sequencing, ultra-low pass whole genome sequencing, and 396-gene targeted panel sequencing, we analyzed 42 plasma-derived ctDNA libraries, representing 4–8 samples per patient with 6–42 days between samples. Integrating tumor fraction, copy number, and somatic variant information, we model tumor clonal dynamics, predict neoantigens, and evaluate consistency of genomic information from orthogonal assays.ResultsWe measured considerable variation in ctDNA tumor faction in each patient, often conflicting with RECIST imaging response metrics. In orthogonal sequencing, we found high concordance between targeted panel and whole exome sequencing in both variant detection and variant allele frequency estimation (specificity = 95.5%, VAF correlation,r= 0.949), Copy number remained generally stable, despite resolution limitations posed by low tumor fraction. Through modeling, we inferred and tracked distinct clonal populations specific to each patient and built phylogenetic trees revealing alterations in hallmark breast cancer drivers, includingTP53, PIK3CA, CDK4, andPTEN. Our modeling revealed varied responses to therapy, with some individuals displaying stable clonal profiles, while others showed signs of substantial expansion or reduction in prevalence, with characteristic alterations of varied literature annotation in relation to the study drug. Finally, we predicted and tracked neoantigen-producing alterations across time, exposing translationally relevant detection patterns.ConclusionsDespite technical challenges arising from low tumor content, metastatic ctDNA monitoring can aid our understanding of response and progression, while minimizing patient risk and discomfort. In this study, we demonstrate the potential for high-frequency monitoring of evolving genomic features, providing an important step toward scalable, translational genomics for clinical decision making.

Background: Genomic mechanisms associated with response to ICI in mTNBC are largely unknown. The aim of this work is to assess the genomic and immune profiles of mTNBC samples collected from patients (pts) treated with ICI. Methods: We identified 31 women with mTNBC treated with ICI (pembrolizumab, n=6, NCT02447003; atezolizumab, n=4, NCT01375842; nivolumab + cabozantinib, n = 6, NCT03316586; pembrolizumab + eribulin, n=8, NCT02513472; atezolizumab + nab-paclitaxel, n=7, NCT01633970) who had tumor tissue or blood available for sequencing obtained before and after ICI. Clinical benefit (CB), here defined as any objective response or stable disease (SD) for > 24 weeks, was observed in 20 pts (65%). An extraordinary responder was defined as having CB ≥ 2 yrs; 5 pts were considered extraordinary responders (range 26-60months). Whole exome sequencing (WES) was done on each tumor and on germline DNA from blood (23 pts had successful WES performed on samples collected before ICI; 5 of these had WES on samples taken after disease progression). RNA sequencing (RNAseq) was successfully performed in 18 of the tumors with WES performed on samples before ICI; and 3 of these had RNAseq on samples taken after disease progression. 18 pts had tumors assessed by multiplex immunofluorescence (mIF) panels encompassing CD4, CD8, PD-1, PD-L1, and cytokeratin on samples collected before ICI. WES, deep targeted panel and low coverage whole genome sequencing were performed on serially collected plasma samples from 22 pts to evaluate tumor fraction and specific mutations. The association between biomarkers and clinical benefit to ICI was assessed. Results: 21 of 31 pts (67%) had received ≥1 prior lines of systemic therapy in the metastatic setting before starting ICI. Among the most frequently mutated genes at baseline are: TP53 (57%); PIK3CA (18%); DNAH5, MYH8 (both 13%); KMT2C, AKT1, LAMA2 (all 9%). Pts with CB had a higher tumor mutational burden (TMB) than pts with no CB (p=0.018). Differential expression analysis of RNAseq data revealed an upregulation of several immune-related genes in pts with CB, indicating increased immune infiltration in that group. Gene set enrichment analysis of this expression data using hallmark and canonical pathway gene sets from MSigDB (nominal p-val < 0.05) showed that, compared to samples from pts without CB, extraordinary responders had elevated transcriptional signatures of several cancer-related pathways associated with cell survival, proliferation and metabolism, as well as genes associated with increased immune infiltration and upregulation of inflammatory response programs. The mIF showed that the tumor microenvironment (TME) of pts with CB were enriched in Cytokeratin-negative/PD-L1-positive cells compared to those without CB (p=0.014). Expression of CD4, CD8 and PD-1 was not significantly different between pts with and without CB. Genomic analysis of circulating tumor DNA, and tumor evolutionary analysis for pts with both pre- and post-ICI samples (acquired resistance) will be presented. Conclusions: Clinical benefit to ICI in mTNBC was associated with upregulation of immune-related pathways, enrichment of non-tumoral PD-L1-positive cells in TME, and high TMB. Citation Format: Romualdo Barroso-Sousa, Juliet Forman, Zachary T. Weber, Katherine Collier, Katrina Z. Kao, Edward T. Richardson, III, Tanya Keenan, Ofir Cohen, Michael P. Manos, Ryan C. Brennick, Patrick Ott, F. Steve Hodi, Deborah A. Dillon, Nancy U. Lin, Eliezer E. Van Allen, Scott Rodig, Eric P. Winer, Elizabeth A. Mittendorf, Catherine J. Wu, Daniel Stover, Nikhil Wagle, Sachet Shukla, Sara Tolaney. Comprehensive genomic analysis reveals molecular correlates of response to immune checkpoint inhibitors (ICI) in metastatic triple-negative breast cancer (mTNBC) [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS4-25.

Introduction: Elevations in serum ferritin and serum iron occur during pediatric extracorporeal membrane oxygenation (ECMO). Previous reports attribute the elevation to frequent red blood cell transfusions and/or hemolysis. Chronic transfusion can cause iron deposition in tissues leading to multisystem organ dysfunction. This study aims identify clinical factors associated with elevated ferritin and iron in pediatric ECMO patients, along with post-decannulation magnetic resonance imaging (MRI) assessment of iron deposition in liver and brain. Methods: Prospective, pilot study, using descriptive statistics to investigate potential associations between patient characteristics, serum ferritin and iron levels, and post-decannulation hepatic and basal ganglia iron deposition. Results: In this study, nine patients (100%) had elevated serum ferritin levels during ECMO. High ferritin levels were more common with veno-arterial than with veno-venous cannulation ( p = 0.026) and were also associated with high plasma free hemoglobin levels ( p < 0.001). Five patients presented with elevated serum iron levels. High serum iron levels were associated with higher daily ( p = 0.016) and cumulative transfusion volumes ( p = 0.013) as well ECMO duration beyond 7 days. MRI scans were performed on three patients with no evidence of abnormal iron deposition detected in the liver or brain. Conclusions: This pilot study shows that during pediatric ECMO, elevations in serum ferritin and serum iron occur and those elevations may be related to the cannulation modality, ECMO duration, amount of hemolysis, and volume of red blood cell transfusions. Further investigation is warranted to fully understand the implications of elevated serum iron and ferritin in pediatric ECMO.

Cell-free DNA (cfDNA) has the potential to inform tumor subtype classification and help guide clinical precision oncology. Here we developed Griffin, a new method for profiling nucleosome protection and accessibility from cfDNA to study the phenotype of tumors using as low as 0.1x coverage whole genome sequencing (WGS) data. Griffin employs a novel GC correction procedure tailored to variable cfDNA fragment sizes, which improves the prediction of chromatin accessibility. Griffin achieved excellent performance for detecting tumor cfDNA in early-stage cancer patients (AUC=0.96). Next, we applied Griffin for the first demonstration of estrogen receptor (ER) subtyping in metastatic breast cancer from cfDNA. We analyzed 254 samples from 139 patients and predicted ER subtype with high performance (AUC=0.89), leading to insights about tumor heterogeneity. In summary, Griffin is a framework for accurate clinical subtyping and can be generalizable to other cancer types for precision oncology applications.

Rationale: Uninvolved normal-appearing airway epithelium has been shown to exhibit specific mutations characteristic of nearby non–small cell lung cancers (NSCLCs). Yet, its somatic mutational landscape in patients with early-stage NSCLC is unknown. Objectives: To comprehensively survey the somatic mutational architecture of the normal airway epithelium in patients with early-stage NSCLC. Methods: Multiregion normal airways, comprising tumor-adjacent small airways, tumor-distant large airways, nasal epithelium and uninvolved normal lung (collectively airway field), matched NSCLCs, and blood cells (n = 498) from 48 patients were interrogated for somatic single-nucleotide variants by deep-targeted DNA sequencing and for chromosomal allelic imbalance events by genome-wide genotype array profiling. Spatiotemporal relationships between the airway field and NSCLCs were assessed by phylogenetic analysis. Measurements and Main Results: Genomic airway field carcinogenesis was observed in 25 cases (52%). The airway field epithelium exhibited a total of 269 somatic mutations in most patients (n = 36) including key drivers that were shared with the NSCLCs. Allele frequencies of these acquired variants were overall higher in NSCLCs. Integrative analysis of single-nucleotide variants and allelic imbalance events revealed driver genes with shared “two-hit” alterations in the airway field (e.g., TP53, KRAS, KEAP1, STK11, and CDKN2A) and those with single hits progressing to two in the NSCLCs (e.g., PIK3CA and NOTCH1). Conclusions: Tumor-adjacent and tumor-distant normal-appearing airway epithelia exhibit somatic driver alterations that undergo selection-driven clonal expansion in NSCLC. These events offer spatiotemporal insights into the development of NSCLC and, thus, potential targets for early treatment.

ObjectiveTo evaluate the discriminatory ability of ultrasound in calcium pyrophosphate deposition disease (CPPD), using microscopic analysis of menisci and knee hyaline cartilage (HC) as reference standard.MethodsConsecutive patients scheduled for knee replacement surgery, due to osteoarthritis (OA), were enrolled. Each patient underwent ultrasound examination of the menisci and HC of the knee, scoring each site for presence/absence of CPPD. Ultrasound signs of inflammation (effusion, synovial proliferation and power Doppler) were assessed semiquantitatively (0–3). The menisci and condyles, retrieved during surgery, were examined microscopically by optical light microscopy and by compensated polarised microscopy. CPPs were scored as present/absent in six different samples from the surface and from the internal part of menisci and cartilage. Ultrasound and microscopic analysis were performed by different operators, blinded to each other’s findings.Results11 researchers from seven countries participated in the study. Of 101 enrolled patients, 68 were included in the analysis. In 38 patients, the surgical specimens were insufficient. The overall diagnostic accuracy of ultrasound for CPPD was of 75%—sensitivity of 91% (range 71%–87% in single sites) and specificity of 59% (range 68%–92%). The best sensitivity and specificity were obtained by assessing in combination by ultrasound the medial meniscus and the medial condyle HC (88% and 76%, respectively). No differences were found between patients with and without CPPD regarding ultrasound signs of inflammation.ConclusionUltrasound demonstrated to be an accurate tool for discriminating CPPD. No differences were found between patents with OA alone and CPPD plus OA regarding inflammation.

Our pilot study tests whether university counseling centers (UCC) can apply the concept of cluster analysis, and geospatial analysis to identify clusters of “hot spots”. Participants: Study partici...

Primary lung tumors are very rare in children and constitute only 0.2 percent of all pediatric malignancies. Carcinoids are the most common primary pediatric lung tumor and account for 80 percent of all primary malignant bronchial tumors. Carcinoid tumors can be histologically categorized as typical or atypical. They are derived from neuroendocrine cells in the bronchial epithelium and are locally infiltrative. Surgical resection of endobronchial carcinoid tumors is the mainstay of treatment with a five-year survival of 95 percent. Endoscopic resection has been reported in adult patients with typical carcinoid tumors (less than 20 mm) with no extrabronchial disease. We present the first pediatric bronchial carcinoid tumor treated with endoscopic resection.

Objective Uterine carcinosarcoma (UCS) is a rare and aggressive form of uterine cancer. It is bi-phasic, exhibiting histological features of both malignant epithelial (carcinoma) and mesenchymal (sarcoma) elements, reflected in ambiguity in accepted treatment guidelines. We sought to study the genomic and transcriptomic profiles of these elements individually to gain further insights into the development of these tumors. Methods We macro-dissected carcinomatous, sarcomatous, and normal tissues from formalin fixed paraffin embedded uterine samples of 10 UCS patients. Single nucleotide polymorphism microarrays, targeted DNA sequencing and whole-transcriptome RNA-sequencing were performed. Somatic chromosomal alterations (SCAs), point mutation and gene expression profiles were compared between carcinomatous and sarcomatous components. Results In addition to TP53, other recurrently mutated genes harboring putative driver or loss-of-function mutations included PTEN, FBXW7, FGFR2, KRAS, PIK3CA and CTNNB1, genes known to be involved in UCS. Intra-patient somatic mutation and SCA profiles were highly similar between paired carcinoma and sarcoma samples. An epithelial-mesenchymal transition (EMT) signature tended to differentiate components, with EMT-like status more common in advanced-stage patients exhibiting higher inter-component SCA heterogeneity. Conclusions From DNA analysis, our results indicate a monoclonal disease origin for this cohort. Yet expression-derived EMT statuses of the carcinomatous and sarcomatous components were often discrepant, and advanced cases displayed greater genomic heterogeneity. Therefore, separately-profiled components of UCS tumors may better inform disease progression or potential.

Certain regions on the surfaces of developing pollen grains exhibit very limited deposition of pollen wall exine. These regions give rise to pollen apertures, which are highly diverse in their patterns and specific for individual species. Arabidopsis thaliana pollen develops three equidistant longitudinal apertures. The precision of aperture formation suggests that, to create them, pollen employs robust mechanisms that generate distinct cellular domains. To identify players involved in this mechanism, we screened natural Arabidopsis accessions and discovered one accession, Martuba, whose apertures form abnormally due to the disruption of the protein kinase D6PKL3. During pollen development, D6PKL3 accumulates at the three plasma membrane domains underlying future aperture sites. Both D6PKL3 localization and aperture formation require kinase activity. Proper D6PKL3 localization is also dependent on a polybasic motif for phosphoinositide interactions, and we identified two phosphoinositides that are specifically enriched at the future aperture sites. The other known aperture factor, INAPERTURATE POLLEN1, fails to aggregate at the aperture sites in d6pkl3 mutants, changes its localization when D6PKL3 is mislocalized, and, in turn, affects D6PKL3 localization. The discovery of aperture factors provides important insights into the mechanisms cells utilize to generate distinct membrane domains, develop cell polarity, and pattern their surfaces.

Purpose: Cancers with short interval between diagnosis and metastasis are associated with aggressive clinical course. Specifically, metastatic relapse of triple-negative breast cancer (TNBC) within 2 years of initial primary diagnosis is associated with marked chemoresistance, rapid progression, and poor prognosis. We hypothesized that rapid relapse TNBCs (rrTNBC; distant metastatic relapse or death 2 years) or no relapse (nrTNBC; no distant relapse or death with at least 5 years follow-up). Patients and Methods: We identified 453 primary TNBCs from three publicly-available datasets and characterized each as rrTNBC, lrTNBC, or nrTNBC. We compiled primary tumor clinical and multi-omic data, including transcriptome (n=453), copy number alterations (CNAs; n=317), and mutations in 171 cancer-related genes (n=317), then calculated expression and immune signatures. Results: Patients with rrTNBC were higher stage at diagnosis (Chi-square p Citation Format: Yiqing Zhang, William Nock, Meghan Wyse, Zachary Weber, Elizabeth J Adams, Asad Sarah, Sinclair Stockard, David Tallman, Jasneet Singh, Junu Bae, Eric P Winer, Nancy U Lin, Yi-Zhou Jiang, Ding Ma, Peng Wang, Leming Shi, Wei Huang, Zhi-Ming Shao, Claire Verschraegen, Mathew Cherian, Maryam B Lustberg, Bhuvana Ramaswamy, Sagar Sardesai, Jeffrey VanDeusen, Nicole Williams, Wesolowski Robert, Daniel G Stover. Machine learning predicts rapid relapse in triple negative breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P4-05-02.

Background 9The OMERACT Ultrasound (US) in calcium pyrophosphate deposition disease (CPPD) sub-task force has been working on the assessment of the utility of US in CPPD since 2014 first creating definitions for CPPD identification and then demonstrating that US is a reliable tool[1]. Objectives Objective of this study is to assess the diagnostic accuracy of US in CPPD Methods This is a multicentre international diagnostic accuracy study involving 17 centres from 9 countries. We enrolled in this study consecutive patients waiting to undergo knee replacement surgery due to severe osteoarthritis. Each patient underwent US examination of the knee, focusing on the menisci and the hyaline cartilage, the day prior to surgery, scoring each site according to the presence/absence of CPP as defined by OMERACT[1]. After surgery, the menisci and the condyles were collected and examined microscopically. Six samples were collected, both from the surface and from the internal part of menisci and cartilage trying to cover a large part of the structure. All slides were observed under transmitted light microscopy and by compensated polarised microscopy. A dichotomous score was given for the presence/absence of CPP crystals. US and microscopic analysis were performed by blinded operators. Sensitivity and specificity of US were calculated using microscopic findings of the menisci and cartilage as the gold standard. Results These preliminary analyses include 30 patients. The mean age was 71yrs (SD±9.1), 19 (63%) were females. 17 patients were positive at US analysis and 12 at microscopic analysis. Diagnostic accuracy results of US at patient level, are presented in figure 1. Conclusion These preliminary results demonstrate that US is a sensitive exam for identification of CPPD with acceptable specificity. US is the first diagnostic technique with consistent reliability and validity to be applied for non-invasive screening for CPPD in clinical practice. References [1] Filippou G, Scire CA, Adinolfi A, et al. Identification of calcium pyrophosphate deposition disease (CPPD) by ultrasound: reliability of the OMERACT definitions in an extended set of joints—an international multiobserver study by the OMERACT Calcium Pyrophosphate Deposition Disease Ultrasound Subtask Force. Ann Rheum Dis 2018;:annrheumdis-2017-212542. doi:10.1136/annrheumdis-2017-212542 Disclosure of Interests Georgios Filippou Speakers bureau: Laborest, Abbvie, BMS, Sanofi, Anna Scanu: None declared, Antonella Adinolfi: None declared, Carmela Toscano: None declared, Raquel Largo-Carazo: None declared, Esperanza Naredo Consultant for: Abbvie, Speakers bureau: AbbVie, Roche, Bristol-Myers Squibb, Pfizer, UCB, Lilly, Novartis, Janssen, and Celgene GmbH, Emilio Calvo: None declared, Gabriel Herrero-Beaumont: None declared, Pascal Zufferey: None declared, Christel Madelaine-Bonjour: None declared, Daryl MacCarter: None declared, Stanley Makman: None declared, Zachary Weber: None declared, Ingrid Moller: None declared, Marwin Gutierrez: None declared, Carlos Pineda: None declared, Denise Clavijo Cornejo: None declared, Hector Garcia: None declared, Victor Ilizaliturri: None declared, Jaime Mendoza Torres: None declared, Raul Pichardo: None declared, Luis Carlos Rodriguez Delgado: None declared, Emilio Filippucci: None declared, Edoardo Cipolletta: None declared, Teodora Serban: None declared, Florentin Ananu Vreju Consultant for: abbvie and novartis, Speakers bureau: abbvie, novartis, pfizer, sandoz, eli lilly, ucb pharma, Gael Mouterde: None declared, Maria-Antonietta d’Agostino: None declared, Marcello Govoni: None declared, Leonardo Punzi Consultant for: BMS, Fidia, Grunenthal, Menarini, Speakers bureau: BMS, Fidia, Grunenthal, Menarini, Nemanja Damjanov Grant/research support from: AbbVie, Pfizer and Roche, Consultant for: Abbvie, Gedeon Richter, Merck, Novartis, Pfizer and Roche., Speakers bureau: Abbvie, Gedeon Richter, Merck, Novartis, Pfizer and Roche., Lene Terslev Speakers bureau: Speakers fee from : Roche, Novartis, Pfizer, MSD, BMS, Celgene, Irene Azzolin: None declared, Giulio Guerrini: None declared, Carlo Alberto Scire: None declared, Annamaria Iagnocco: None declared

PurposeMetastatic relapse of triple-negative breast cancer (TNBC) within 2 years of diagnosis is associated with particularly aggressive disease and a distinct clinical course relative to TNBCs that relapse beyond 2 years. We hypothesized that rapid relapse TNBCs (rrTNBC; metastatic relapse or death 2 years).Patients and MethodsWe identified 453 primary TNBCs from three publicly-available datasets and characterized each as rrTNBc, lrTNBC, or ‘no relapse’ (nrTNBC: no relapse/death with at least 5 years follow-up). We compiled primary tumor clinical and multi-omic data, including transcriptome (n=453), copy number alterations (CNAs; n=317), and mutations in 171 cancer-related genes (n=317), then calculated published gene expression and immune signatures.ResultsPatients with rrTNBC were higher stage at diagnosis (Chi-square pTP53mutations were significantly more frequent in rrTNBC compared to lrTNBC (Fisher exact FDR p=0.009). To develop an optimal classifier, we used 77 significant clinical and ‘omic features to evaluate six modeling approaches encompassing simple, machine learning, and artificial neural network (ANN). Support vector machine outperformed other models with average receiver-operator characteristic area under curve >0.75.ConclusionsWe provide a new approach to define TNBCs based on timing of relapse. We identify distinct clinical and genomic features that can be incorporated into machine learning models to predict rapid relapse of TNBC.

1025 Background: Analysis of circulating tumor DNA (ctDNA) over time allows non-invasive evaluation of tumor genomic evolution. We characterize changes in tumor fraction (TFx), somatic copy number alterations (SCNAs), and somatic mutations (muts) over time in patients (pts) with BRCA1/2 muts and metastatic breast cancer (mBC) who received a PARP inhibitor (PARPi) or platinum chemotherapy. Specifically, we seek to identify the frequency of BRCA1/2 reversion muts. Methods: Pts with mBC and germline or somatic BRCA1/2 muts were identified on a banking protocol of prospectively-collected serial samples of blood and plasma. Control pts without a BRCA1/2 mut were matched 2:1 by age and hormone receptor (HR) status. Ultra-low-pass whole genome sequencing (ULPWGS) with 0.1x depth was performed on all plasma samples (n = 103) and the ichorCNA algorithm was used to determine TFx and SCNAs. Targeted panel sequencing (TPS) of 402 cancer-related genes was performed at 10,000x depth on plasma samples, and one blood sample per pt. The panel includes BRCA1/2 and 38 other DNA damage repair (DDR) genes. Somatic muts were identified by joint calling with Mutect2 across plasma timepoints with paired pt normal blood. Germline variant calling from TPS on blood with HaplotypeCaller was used to confirm germline muts in BRCA1/2.Results: We identified 10 pts with mBC with a germline (n = 7) or somatic (n = 3) BRCA1 (n = 2) or BRCA2 (n = 8) mut and banked blood and plasma samples at 3-9 timepoints at a median of 8 weeks apart (range 1-43). The control cohort of 20 pts with mBC and wildtype BRCA1/2 was well matched by age and HR status. All pts with BRCA1/2 muts received a PARPi and/or platinum chemotherapy at some point during sample collection. Half of control pts received platinum chemotherapy. Germline BRCA1/2 muts were confirmed in all 7 pts with known germline muts. Among the BRCA1/2 mut cohort, median TFx was 0.04 (range 0-0.57) with 20% of samples having TFx > 0.10. A median of 1.5 (range 0-39) somatic muts per pt were found in DDR genes. Four pts (40%) had secondary non-reversion muts in BRCA1/2. A reversion mut of a germline BRCA2 mut, restoring the open reading frame of BRCA2, was discovered at the last timepoint from 1 pt while receiving carboplatin. A germline BRCA1/2 reversion mut in this cohort occurred in 2.3% of samples, 14.3% of pts. There was no significant difference in the percent of genome with a SCNA between the first and last time point, nor before and after PARPi/platinum. The somatic mut landscape and clonal evolution of TPS using PyClone will be presented. Conclusions: Evaluation of serial ctDNA samples for TFx, SCNAs, and somatic muts from banked plasma and blood from pts with mBC is feasible. The frequency of reversion muts in BRCA1/2 was low, suggesting that either their incidence is low or ctDNA TPS is not sensitive enough to detect them. Secondary non-reversion muts in BRCA1/2 and other somatic DDR muts were more common. SCNAs were stable over time.

Introduction: Circulating tumor DNA (ctDNA) offers the ability to repeatedly interrogate tumor genomic information, providing an opportunity for real-time monitoring of tumor genomic dynamics. In this study, we deeply analyzed multiple ctDNA samples collected over narrow time frames (days-to-weeks) from seven patients with metastatic triple-negative breast cancer (mTNBC), a cancer type known to have high ctDNA content. Methods: Patients with mTNBC were enrolled in a clinical trial of multi-kinase inhibitor cabozantinib, providing uniform and targeted treatment, and samples were collected day 1, day 8, then every 21 to 42 days on study. ctDNA was extracted from each plasma sample and underwent ultra-low pass whole genome sequencing (ULP-WGS; average depth 0.1x; n=42 samples), deep targeted panel sequencing (TPS) of 402 cancer-related genes with unique molecular identifier indexing (depth 10,000x; n=42 samples), and samples with tumor fraction (TFx) >10% underwent whole exome sequencing (WES; depth 200x; n=31 samples), with whole blood germline sequencing of both TPS and WES for subsequent analyses. Somatic copy number alterations (SCNAs) were identified from ULP-WGS and WES. PyClone with TPS was employed for clonal dynamic analyses. Predicted neoantigens were determined from WES using HLAthena. Results: A total of 42 total plasma samples from 7 patients (range 4-8 samples per patient) were collected at narrow time intervals, median 21 days (range 6 to 42 days) between samples. The median TFx across all samples was 18.1% (range 2.5% to 44.3%). TFx estimates were concordant when comparing orthogonal sequencing approaches (ULP-WGS, WES) and tumor fraction estimation algorithms (ichorCNA, FACETS). Despite all seven patients having ‘stable disease’ as best objective response, TFx dynamics were widely variable with TFx declining to lower limit of detection in three of seven patients. Of all samples, 31/42 (73.8%) had tumor fraction >10% and underwent WES; each patient had at least 3 samples that underwent both WES and TPS. There was strong agreement between TPS and WES: across all 31 shared samples, mutation recall in TPS versus WES (gold standard) was 95.5%. Variant allele frequency across all mutations detected in both TPS and WES was highly concordant (Pearson’s r=0.949). Clonal mutations were consistently detected across multiple samples within patients. When comparing genome-wide copy number from last to first available sample within each patient, copy number log ratios were largely stable within patients (union Pearson’s r=0.924) and there were not recurrent shifts in SCNAs across patients. Through statistical modeling of TPS data, we tracked distinct clonal populations for each patient over their sampling windows. Modeled clonal architecture in most patients revealed stable, polyclonal profiles, with important breast cancer driver alterations (e.g. TP53 and PIK3CA) recurrently presenting at high prevalence. Infrequently, we also detected emergence and expansion of clones over narrow time frames (weeks) containing acquired alterations poorly annotated in the breast cancer literature. We successfully predicted neoantigens from ctDNA WES at multiple time points in each patient, with evidence that patients acquired new mutations predicted to be ‘strong binder’ neoantigens over time on therapy. Conclusions: Analysis of serial ctDNA samples collected at narrow time intervals (days-to-weeks) provides unique insight into the dynamics of ctDNA. We demonstrate strong concordance across ctDNA sequencing appraoches. Evolving genomic features of tumor populations can be identified via ctDNA while on treatment, potentially providing real time insight for clinical decision-making. Citation Format: Daniel G Stover, Katharine A Collier, David Tallman, Juliet Forman, Sachet Shukla, Sarah Asad, Justin Rhoades, Samuel Freeman, Mathew Cherian, Sagar Sardesai, Romualdo Barroso-Sousa, Carrie Cibulskis, Niall Lennon, Gavin Ha, Sara M Tolaney, Viktor A Adalsteinsson, Zachary Weber. Modeling clonal structure over narrow time frames via circulating tumor DNA in metastatic breast cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PD9-08.

Visually normal cells adjacent to, and extending from, tumors of the lung may carry molecular alterations characteristics of the tumor itself, an effect referred to as airway field of cancerization. This airway field has been postulated as a model for early events in lung cancer pathogenesis. Yet the genomic landscape of somatically acquired molecular alterations in airway epithelia of lung cancer patients has remained unknown. To begin to fill this void, we sought to comprehensively characterize the genomic architecture of chromosomal alterations inducing allelic imbalance (AI) in the airway field of the most common type of lung tumors, non–small cell lung cancer (NSCLC). To do so, we conducted a genome-wide survey of multiple spatially distributed normal-appearing airways, multiregion tumor specimens, and uninvolved normal tissues or blood from 45 patients with early-stage NSCLC. We detected alterations in airway epithelia from 22 patients, with an increased frequency in NSCLCs of squamous histology. Our data also indicated a spatial gradient of AI in samples at closer proximity to the NSCLC. Chromosome 9 displayed the highest levels of AI and comprised recurrent independent events. Furthermore, the airway field AI included oncogenic gains and tumor suppressor losses in known NSCLC drivers. Our results demonstrate that genome-wide AI is common in the airway field of cancerization, providing insights into early events in the pathogenesis of NSCLC that may comprise targets for early treatment and chemoprevention. Cancer Res; 76(13); 3676–83. ©2016 AACR.

Copy number aberrations (CNAs) are gains and losses of large genomic segments present across most cancer types and are a hallmark of cancer genomic alterations. However, the processes underlying CNAs and characteristic patterns of CNAs are poorly understood. Using single nucleotide variant (SNV) data, bioinformatic advances have identified underlying mutational signatures resulting from distinct mutational processes. Mutational signatures have led to a variety of discoveries, several of which are being investigated in clinical management of cancer. The development of algorithms able to uncover similar signatures for CNAs, rather than SNVs, is still in its infancy. Here we present an analysis package for the R programming language called CNSigs that allows for the robust and reproducible derivation of copy number signatures. Based on a list of extracted copy number features previously verified in ovarian cancer, we utilize mixed model approaches and non-negative matrix factorization to derive CNA signatures across cancer types. The development of a package to derive signatures from copy number data allows further investigation of underlying processes that may be responsible for these CNA fingerprints. To verify the reproducibility of the signatures, we derived signatures from two independent breast cancer datasets that use distinct copy number segmentation approaches. From these independent datasets we were able to recover the same signatures with high accuracy. We identified five signatures that are distinct from known breast cancer receptor-based or expression-based subtypes, yet reveal unique associations with underlying mutations, mutational processes, and transcriptional programs. To validate robustness, we applied the pipeline to 11 different cancer types from the TCGA dataset and showed that we were able to derive signatures from all of these cancer types of varying sample sizes. We identified certain signatures that cut across tumor types, while others are distinct to individual cancers. This work lays the groundwork for further analysis into the underlying molecular processes leading to these copy number signatures seen across cancer types. The CNSigs package also allows researchers to easily analyze their own samples to look for signatures in their copy number profiles and to compare these to signatures previously derived for their cancer type. Citation Format: David Tallman, Sinclair Stockard, Zachary T. Weber, Sarah Asad, Katharine Collier, Elizabeth Adams, Jerome Bey, Daniel G. Stover. CNSigs: An R package for the identification of copy number signatures [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5471.

Introduction: Circulating tumor DNA (ctDNA) offers the ability to serially interrogate tumor genomic information, providing an opportunity for real-time monitoring of tumor genomic shifts. In this study, we deeply analyzed multiple ctDNA samples collected over narrow time frames (days-to-weeks) from seven patients with metastatic triple-negative breast cancer (mTNBC), a cancer type known to have high ctDNA content. Methods: Patients were enrolled in a clinical trial of the multikinase inhibitor cabozantinib, providing uniform and targeted treatment, and samples were collected at study entry, then every 21 to 42 days on study. ctDNA was extracted from each plasma sample and underwent ultra-low pass whole-genome sequencing (ULP-WGS; average depth 0.1x; n=42 samples), deep targeted panel sequencing of 402 cancer-related genes with unique molecular identifier indexing (depth 10,000x; n=42 samples), and samples with tumor fraction >10% underwent whole-exome sequencing (WES; depth 200x; n=31 samples), with germline sequencing of both targeted panel and WES for downstream analyses. SCNAs were identified from ULP-WGS and WES. PyClone with targeted panel sequencing data was employed for clonal evolution analyses. Predicted neoantigens were determined from WES. Results: 42 total plasma samples were analyzed (range 4-8 samples per patient) collected at narrow time intervals, ranging from 6 to 42 days (median 21 days) between samples. The median tumor fraction across all samples was 18.1% (range 2.5% to 44.3%). Tumor fraction/purity estimates were largely concordant when comparing orthogonal sequencing approaches (ULP-WGS, WES) and tumor fraction estimation algorithms (ichorCNA and FACETS, respectively). Of all samples, 31/42 (73.8%) had tumor fraction >10% and underwent WES; each patient had at least 3 samples that underwent WES. SCNAs were largely concordant between ULP-WGS and WES (all Cohen’s kappa >0.8). When comparing first and last available sample, there were not recurrent shifts in SCNAs. At lower tumor fractions, fewer mutations were detected, yet clonal alterations were consistently identified across multiple samples within patients. Through statistical modeling, we tracked distinct clonal populations for each patient and found diverse clonal architectures and dynamics through treatment. Additionally, several emergent somatic alterations were discovered at late time points, including alterations found in coding regions, proximal regulatory sites, and introns of key drug targets, underscoring the speed at which tumors can adapt to therapeutic agents. Conclusions: Analysis of serial ctDNA samples collected at narrow time intervals (days-to-weeks) provides unique insight into the dynamics of ctDNA as well as clonal evolution. We show that despite low tumor content, evolving genomic features of tumor populations can be identified while on treatment, potentially providing real-time insight for clinical decision-making. This abstract is also being presented as Poster A27. Citation Format: Zachary Weber, David Tallman, Sinclair Stockaard, Katharine Collier, Sarah Asad, Justin Rhoades, Samuel Freeman, Heather A. Parsons, Sara M. Tolaney, Gavin Ha, Viktor A. Adalsteinsson, Daniel G. Stover. Clonal evolution over narrow time frames via circulating tumor DNA in metastatic breast cancer [abstract]. In: Proceedings of the AACR Special Conference on Advances in Liquid Biopsies; Jan 13-16, 2020; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(11_Suppl):Abstract nr PR06.

Background:The OMERACT Ultrasound (US) in calcium pyrophosphate deposition disease (CPPD) sub-task force has been working on the use of US in CPPD since 2014 first creating definitions for CPPD identification and then assessing the reliability[1].Objectives:Objective of this study is to assess the diagnostic accuracy (truth) of US in CPPD.Methods:Consecutive patients waiting to undergo knee replacement surgery due to osteoarthritis were enrolled in 12 centres from 6 countries. Each patient underwent US examination of the knee, focusing on the menisci and the hyaline cartilage, the day prior to surgery, scoring each site for presence/absence of CPP as defined previously[1]. After surgery, the menisci and the condyles were retrieved and examined microscopically. Six samples were collected, both from the surface and from the internal part of menisci and cartilage trying to cover a large part of it. All slides were observed under transmitted light microscopy and by compensated polarised microscopy. A dichotomous score was given for the presence/absence of CPP. US and microscopic analysis were performed by different operators, blind to each other’s findings. Sensitivity and specificity of US were calculated using microscopic findings as the gold standard.Results:101 patients have been enrolled in the study. 33 patients have been excluded due to loss of anatomical pieces at surgery. The mean age of the remaining 68 pts was 71yo (±8), 44 women, 34 were affected by CPPD according to microscopy. Overall and per site diagnostic US accuracy results are presented in table 1Diagnostic accuracySensitivitySpecificityPositive Predictive valueNegative Predictive valueGlobal0.750.910.590.690.87Medial meniscus0.820.870.770.770.87Lateral meniscus0.750.830.680.680.83Medial cartilage0.860.790.920.880.85Lateral cartilage0.820.710.880.770.84Medial side (combined cartilage and meniscus)0.820.880.760.790.87Lateral side (combined cartilage and meniscus)0.780.880.690.730.86Conclusion:Our results demonstrate that US is an accurate exam for identification of CPPD. The best combination of sensitivity and specificity is achieved by examining the medial aspect of the knee.References:[1]Filippou G, Scirè CA, Adinolfi A,et al.Identification of calcium pyrophosphate deposition disease (CPPD) by ultrasound: reliability of the OMERACT definitions in an extended set of joints—an international multiobserver study by the OMERACT Calcium Pyrophosphate Deposition Disease Ultrasound Subtask Force.Ann Rheum Dis2018;:annrheumdis-2017-212542. doi:10.1136/annrheumdis-2017-212542Disclosure of Interests:Georgios Filippou: None declared, Anna Scanu: None declared, Antonella Adinolfi: None declared, Carmela Toscano: None declared, Dario Gambera: None declared, Raquel Largo: None declared, Esperanza Naredo: None declared, Emilio Calvo: None declared, Gabriel Herrero-Beaumont: None declared, Pascal Zufferey: None declared, Christel Madelaine-Bonjour: None declared, Daryl MacCarter: None declared, Stanley Makman: None declared, Zachary Weber: None declared, Fabiana Figus: None declared, Ingrid Möller: None declared, Marwin Gutierrez: None declared, Carlos Pineda: None declared, Denise Clavijo Cornejo: None declared, Héctor García: None declared, Victor Ilizaliturri: None declared, Jaime Mendoza Torres: None declared, Raul Pichardo: None declared, Luis Carlos Rodriguez Delgado: None declared, Emilio Filippucci Speakers bureau: Dr. Filippucci reports personal fees from AbbVie, personal fees from Bristol-Myers Squibb, personal fees from Celgene, personal fees from Roche, personal fees from Union Chimique Belge Pharma, personal fees from Pfizer, outside the submitted work., Edoardo Cipolletta: None declared, Teodora Serban: None declared, Catalin Cirstoiu: None declared, Florentin Ananu Vreju: None declared, Dun Grecu: None declared, Gael Mouterde: None declared, Marcello Govoni: None declared, Leonardo Punzi: None declared, Nemanja Damjanov Grant/research support from: from AbbVie, Pfizer, and Roche, Consultant of: AbbVie, Gedeon Richter, Merck, Novartis, Pfizer, and Roche, Speakers bureau: AbbVie, Gedeon Richter, Merck, Novartis, Pfizer, and Roche, Lene Terslev Speakers bureau: LT declares speakers fees from Roche, MSD, BMS, Pfizer, AbbVie, Novartis, and Janssen., Carlo Alberto Scirè: None declared, Annamaria Iagnocco Grant/research support from: Abbvie, MSD and Alfasigma, Consultant of: AbbVie, Abiogen, Alfasigma, Biogen, BMS, Celgene, Eli-Lilly, Janssen, MSD, Novartis, Sanofi and Sanofi Genzyme, Speakers bureau: AbbVie, Alfasigma, BMS, Eli-Lilly, Janssen, MSD, Novartis, Sanofi

Background Lower respiratory tract infection (LRTI) is the leading cause of pediatric hospitalizations in the United States, with significant morbidity and mortality. Native American children are at increased risk for severe illness during LRTI. Yet, the reasons for this increased risk are poorly understood. Socio-economic status and/or a genetic predisposition have been postulated as possible causes. In addition, the spectrum of LRTI presentations has not been adequately described in this patient population. The objective of this study was to define the clinical presentations of LRTI and highlight the differences between Native American and non-native American previously healthy patients under the age of 24 months. Methods We performed a retrospective chart review during the 2017–2018 respiratory season. We reviewed 357 medical records, and included 192 patients in the analysis that were full term, previously healthy, and met our inclusion criteria. We recorded demographic information, clinical and laboratory data, and outcomes. Results Of 192 patients, 39 were Native American and 153 were non-native American. We found no differences in gestational age, gender or age (median age was 5 and 7 months, respectively) between groups. We found no difference in rates of vaccination, upper respiratory symptoms, cough, wheezing, crackles, increased work of breathing or peripheral white blood cell count at presentation. In addition, we found no differences in antibacterial use or length of antimicrobial therapy during hospitalization. Native American children had a statistically significant higher length of hospitalization (P = 0.01) as well as days of oxygen supplementation (mean 4.9 vs. 3 days; P = 0.006) compared with non-native Americans. Furthermore, Native American children had a significantly higher percentage of PICU admissions (28% vs. 10.4%; P = 0.008) as well as intubation rate (26% vs. 8%; P = 0.04) compared with non-native Americans. Conclusion Native American children had increased length of hospitalization associated with severe illness including longer oxygen supplementation, higher PICU admission rate and need for mechanical ventilatory support. Disclosures All authors: No reported disclosures.

A term female infant (39+4 weeks) with a birthweight of 3,055 g is born via spontaneous delivery to a gravida 4, para 3 woman. Maternal history is significant for Ashkenazi Jewish heritage and known connective tissue disease requiring plaquenil. Maternal serologies and prenatal testing are unremarkable with the exception of herpes simplex virus (HSV) 1 immunoglobulin (Ig) G being positive and IgM inconclusive. The mother was not taking antiviral therapy during pregnancy or at delivery. On admission to the labor and delivery department, the infant’s mother is without prodromal symptoms or genital lesions concerning for HSV. The patient is born via spontaneous vaginal delivery through meconium-stained amniotic fluids, with Apgar scores of 8 and 9 at 1 and 5 minutes, respectively. At the time of delivery, the infant has normal physical examination findings and is kept in the mother-baby unit for routine newborn care. At about 30 hours after birth, the newborn team is called to the bedside by the nurses because of the development of vesicular lesions on the oral mucosa of the lower lip (Fig). On examination of the oral mucosa, multiple vesicular lesions are noted, which measure 1 to 2 mm in size. Because of …

BACKGROUND: There are very few strategies to treat non-small cell lung cancer (NSCLC) at its primitive stages largely due to our poor understanding of molecular aberrations in development of the malignancy and that would be ideal targets for early treatment. Work from our group and others revealed that visually "normal" airway cells carry alterations (e.g. expression and copy number changes) that are characteristic of the nearby lung tumor, signifying a "field of cancerization" that is pertinent to the pathobiology of the tumor. Yet, the landscape of driver alterations in the cancerization field of the normal-appearing airway remains largely unexplored. METHODS: 409 cancer-associated genes were surveyed by deep targeted sequencing in tumor adjacent (small airways) and distant (mainstem bronchi/large airways, nasal epithelia, normal lung parenchyma) normal-appearing tissues as well as in multi-region tissues from paired tumors contrasting events in blood, for a total of 500 samples from 48 patients with early-stage NSCLC (11 squamous cell carcinomas and 37 adenocarcinomas). Somatic point mutations were interrogated by aggregating multiple mutation callers. Mutation were then paired with our recent study of genome-wide copy number alterations (CNAs) inferred from high-density whole-genome SNP microarrays. RESULTS: We identified somatic point mutations in 76 normal-appearing field samples (75% of patients) which were overall concordant with profiles in corresponding NSCLCs. Somatic mutation signatures associated with smoking, APOBEC activity and DNA mismatch repair were common to both airway field samples and NSCLCs from smoker cases. Airway field mutation burdens, while varied, increased significantly with proximity to the tumor. Most field mutations were observed in tumor-adjacent small airways (from 31 patients) some of which were in known drivers such as TP53, KEAP1, STK11 and KRAS. Somatic mutations were also identified in more distant normal samples including large airways (TP53, SETD2, CDKN2A), normal lung parenchyma (RB1, RET) and nasal epithelium (AKT1). We then integrated point mutation and CNA data and found "two-hit" gene alteration patterns in airway field samples that were consistent with their matched NSCLCs, including point mutated gene/CNA pairs such as KEAP1 and STK11/19p loss, TP53/17p loss and KRAS/12p gain. Some additional cases exhibited single hits (e.g., TP53) in their airway field progressing to "two-hits" (e.g., TP53/17p loss) in the matched NSCLC. CONCLUSIONS: Our integrative high-throughput sequence and genome-wide SNP analyses implicate early mutational processes and putative drivers in the progression of the mutagenized "normal" airway field of cancerization to NSCLC thus offering insights into strategies for interception via early detection and personalized prevention. Citation Format: Smruthy Sivakumar, Yasminka Jakubek, F Anthony San Lucas, Wenhua Lang, Christina McDowell, Zachary Weber, Carmen Behrens, Neda Kalhor, Cesar Moran, Randa El-Zein, Gareth Davies, Junya Fujimoto, Reza Mehran, Stephen G. Swisher, Jing Wang, Jerry Fowler, Steven Dubinett, Avrum E. Spira, Erik Ehli, Ignacio I. Wistuba, Paul Scheet, Humam Kadara. Somatic mutational processes in the cancerization field of the normal-appearing airway reveal early drivers in the development of non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3997.

Transcriptome sequencing (mRNA-seq) is becoming a very versatile technique for profiling tumors, extending beyond its original intent of transcript quantification, identification of alternative transcripts, and detection of gene fusions. For instance, through recent advancements in RNA-seq data analyses, one can now computationally assess allele-specific gene expression and generate profiles of expressed somatic mutations. Here, we demonstrate the ability to identify chromosomal allelic imbalances (AI) through detection of haplotype-specific patterns in gene transcripts. This class of RNA-based observations may potentially reveal DNA-level chromosomal allelic imbalances or uncover large regions of transcription deregulation. From the TCGA Uterine Carcinosarcoma project, we downloaded exome and RNA sequencing data for 48 patients’ tumor/normal sample pairs in addition to their clinical annotations. We also downloaded Affymetrix SNP6-based DNA copy number event calls made by the TCGA for use as a gold standard when evaluating the AI calls in the exome and RNA-seq. AI calls were made in both the exome and RNA-seq data by: (1) calling 1000 Genomes genotypes in the sequencing data, (2) phasing haplotypes and then (3) characterizing haplotype imbalances using a tool that we developed called hapLOHseq. hapLOHseq applied to the exome and RNA-seq data both resulted in a 72% specificity for identifying the gold standard AI events. In RNA-seq data we detected 43% of the chromosomal AI events identified in the exome sequencing data. When considering AI events specifically detected in the RNA-seq and not the gold standard (RNA-specific AI), the data suggest that higher RNA-specific AI loads could be negatively associated with survival (p-val = 0.076), with higher RNA-specific AI load patients having a median survival of 771 days compared to 1526 days for those patients with lower loads of RNA-specific AI. In conclusion, our results suggest that analysis of chromosomal AI in RNA-seq has equal specificity for detecting DNA-level AI when compared to exome sequencing, although at lower sensitivities. Clinically, our analyses suggest that patients with higher RNA-specific AI load may have a worse overall survival prognosis. The AI we are identifying in the RNA-seq samples may reflect large-scale transcription defects, resulting in a negative impact on the survival of patients. One possible cause of RNA-specific allelic imbalance could be the presence of cis mutations that impact a large-region of the transcription of one of the two haplotypes. Currently, we are identifying areas for improvement in our analytical methods, while interrogating and characterizing exome and RNA-seq AI in additional data sets. Citation Format: Francis A. San Lucas, Yihua Liu, Zachary Weber, Erik Ehli, Gareth Davies, Paul Scheet. Characterization of chromosomal allelic imbalances through RNA-seq [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3572. doi:10.1158/1538-7445.AM2017-3572

Lung cancer, of which non-small cell lung cancer (NSCLC) is the most common form, is the second most prevalent cancer in the U.S. and the leading cause of cancer mortality. Field cancerization phenomenon establishes that normal appearing tissues surrounding a tumor will exhibit a field effect – particularly in smokers – with the tissues proximal to the tumor showing more, and even sharing, alterations with the tumor. Previous studies in NSCLC provide evidence for both complex progression trajectories, yet, precise mechanisms remain unknown. To investigate the field cancerization phenomenon, we conducted a genome-wide survey of acquired DNA alterations in normal-appearing tissues including small airways adjacent to NSCLC, mainstem bronchi (large airways), nasal epithelium, distant normal lung and blood, along with multiple samples from their surgically resected paired tumors, for a total of 500 samples from 48 patients with early-stage NSCLC (11 squamous cell carcinomas and 37 adenocarcinomas). Since we expect low mutation burdens and mutant cell fractions in pathologically normal tissues, we assessed somatic point mutations via deep targeted sequencing of 409 genes and paired these data with our recent study of acquired copy number alterations (CNAs) inferred from high-density whole-genome SNP microarrays, thus offering a deep and wide survey of somatic DNA alterations. After aggregating results from multiple mutation callers, we observed somatic mutations (exonic, splicing and UTRs) in 257 samples of which 65 (in 35 patients) were from normal-appearing field (non-tumor, non-blood) samples. Tumor and field samples in smokers showed a higher mutation burden and large proportion of C:G>A:T changes. We also observed concordance of mutations in the airway and corresponding tumor profiles. Further, a statistically significant field effect was established with mutational burden (measured by the variant allele frequency) increasing with proximity to the tumor. Among the 35 NSCLC cases with field mutations, the small adjacent (to tumor) airways in six patients exhibited mutations in lung cancer drivers such as KRAS, STK11, TP53 and KEAP1. We also identified mutations in the large airway (TP53, SETD2, CDKN2A), distant normal lung parenchyma (RB1, RET) and nasal epithelium (AKT1). We then correlated point mutation and CNA mutation profiles. Of eight cases showing a nonsynonymous or stopgain variants in established lung cancer drivers in the airways, four exhibited putative “two-hit” progression models, e.g. both KEAP1 and STK11 mutations with 19p loss, overlapping TP53 mutation/17p loss, or KRAS mutation/12p gain. Our findings in normal-appearing tissues of the respiratory epithelium offer insights into the earliest mutational events in their progression to NSCLC and, possibly, in tumor relapse and that may represent suitable targets for early detection and chemoprevention. Citation Format: Smruthy Sivakumar, Yasminka Jakubek, Wenhua Lang, Tina McDowell, Melinda M. Garcia, Chi-Wan Chow, Zachary Weber, Carmen Behrens, Neda Kalhor, Cesar Moran, Randa El-Zein, Gareth Davies, Junya Fujimoto, Reza Mehran, Stephen G. Swisher, Jing Wang, Avrum E. Spira, Jerry Fowler, F Anthony San Lucas, Ignacio I. Wistuba, Erik Ehli, Paul Scheet, Humam Kadara. Mutational landscape in the normal-appearing airway cancerization field of early-stage non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1434. doi:10.1158/1538-7445.AM2017-1434

Uterine carcinosarcoma (UCS), also known as Malignant Mixed Mullerian Tumor, is a neoplasm of the female genital tract that shows histological features of both carcinoma and sarcoma. Some studies have indicated that UCS arises from sarcomatous differentiation of high-grade carcinoma while others have confirmed a bi-clonal nature of the tumor. Given these differences, further investigation in the origin of this tumor with newer approaches and technologies is warranted. In this study we determined the allelic imbalance (AI) profiles of the two components of UCS and compared the AI events that were shared by, or differed between, them. Samples were obtained from 10 patients diagnosed with UCS and were preserved in formalin fixed paraffin embedded (FFPE) blocks. The two components (carcinoma & sarcoma) were identified and micro-dissected, and whole-genome DNA micro-arrays were applied to the isolated DNA. We then applied a sensitive computational method that combines haplotype information with SNP array data to identify somatic segmental copy number variations and copy-neutral loss of heterozygosity (cnLOH). After we obtained the AI events we filtered small events ( Citation Format: Aditya S. Deshpande, Zachary Weber, Raed Sulaiman, Natasha Flier, Cheryl Ageton, Mary Fagerness, Joseph Sulaiman, Gareth E. Davies, David Starks, Luis Rojas-Espaillat, Paul A. Scheet, Erik Ehli. Allelic imbalance analysis of uterine carcinosarcoma: An inquiry into the dual nature of the neoplasm. [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 131.

We evaluated the clinical relevance of catenins, cadherins and cell cycle regulators in stage IV or recurrent endometrial carcinoma in a multi-center phase II trial (GOG protocol #119).Tissue microarrays of metastatic or recurrent (n=42) tumor were developed and immunohistochemistry was performed. Average expression (percent staining x intensity) was assessed in tumor epithelium ((E)) and stroma ((S)) and categorized into tertiles (T1, T2, T3) for E-cadherin(E), N-cadherin(E), alpha-catenin(E), beta-catenin(E), gamma-catenin(E), p120-catenin(E) and Ki-67(E); as negative, below median or above median for p16(E), p27(E) and CD44(S); or as negative or positive for p53(E), Ki-67(S) and APC(S) (adenomatous polyposis coli). End points included response and survival.E-cadherin(E), p16(E), and p53(E) varied by race (p=0.003, p=0.024, p=0.002,) and N-cadherin(E), Ki-67(E), p16(E) and p27(E) by tumor type (p=0.015, p=0.011, p=0.005, p=0.021). Correlations were observed among E-cadherin(E) with p120(E) (r=0.66), p53(E) (r=-0.32), alpha-catenin(E) (r=0.52), beta-catenin(E) (r=0.58), and gamma-catenin(E) (r=0.58). High E-cadherin(E) (T2 or T3) versus low (T1) expression was associated with better survival in unadjusted (hazard ratio [HR]=0.14, 95% confidence interval [CI]=0.06-0.37 or HR=0.17, 95% CI=0.07-0.42) and adjusted models (HR=0.18, 95% CI=0.05-0.59 or HR=0.22, 95% CI=0.07-0.70). High p16(E) versus negative expression was associated with worse survival in unadjusted (HR=3.87, 95% CI=1.74-8.61) and adjusted (HR=4.18, 95% CI=1.28-13.6) models. Positive versus negative expression of p53(E) was associated with worse survival in unadjusted (HR=2.31, 95% CI=1.16-4.60) but not adjusted models.E-cadherin(E) and p16(E) appear to be clinically relevant, independent prognostic factors in stage IV or recurrent endometrial cancers treated with Tamoxifen and Medroxyprogesterone acetate, and merit further study.

Background Endoscopic submucosal dissection (ESD) increases en bloc and histologically complete resection rate of neoplastic mucosal tumors but is technically more demanding than EMR. Limited data are available comparing the efficacy and safety of a new ESD designed to overcome these limitations and conventional ESD (C-ESD) techniques. Objective To compare the safety, efficacy, and operation time of the new HybridKnife ESD (HK-ESD) with C-ESD in the esophagus. Design Prospective, randomized, controlled study. Setting Animal research laboratory. Subjects Seventeen anesthetized Yorkshire pigs. Interventions Removal of a 4-cm length of half-circumference esophageal mucosa by C-ESD with Hook knife or Flexknife versus HK-ESD. Main Outcome Measurements Procedure time, en bloc and complete resection rate, and complications (bleeding and perforation). Results All resections were completed en bloc. Procedure time was shorter in C-ESD. However, it was similar after 12 procedures. Significantly more bleeding occurred during C-ESD (28 vs 12, P = .0007). Histological muscularis propria injuries occurred with equal frequency (16 vs 17) and were mostly seen during the first 11 procedures. There were 3 perforations (2 endoscopic, 1 histological), all with C-ESD. Limitations Nonsurvival study, use of 2 conventional knives, no training period for a new procedure. Conclusions The HK-ESD technique was equally effective as the C-ESD technique for successful en bloc resection and was safer with less bleeding and perforation. Although procedure time was longer in HK-ESD, the difference became nonsignificant after 12 procedures.

Context: Ten to 30% of patients with papillary thyroid cancer (PTC) develop recurrent disease and may benefit from innovative adjuvant therapies. Immune-based therapies are under investigation to treat many types of cancer. The role of the immune system in PTC is poorly understood. Objective: We investigated whether tumor-associated lymphocytes (TAL), in the absence of background thyroiditis (LT), contribute to disease severity. We hypothesized that the type of lymphocytes associated with PTC would correlate with parameters of disease. Design: This retrospective study analyzed archived PTC samples for the presence of TAL and/or LT. A group of patients with TAL was evaluated for lymphocyte subsets by immunohistofluorescence. Patients and Setting: One hundred PTC patients were analyzed for LT and TAL, and 10 PTC patients with TAL were assessed for lymphocyte subsets at University of Colorado Hospital. Main Outcome: We assessed correlations between disease and the presence of TAL, LT, and lymphocyte subset frequency. Results: Patients with TAL exhibited higher disease stage and increased incidence of invasion and lymph node metastasis compared with patients without lymphocytes or with LT. CD4+ T cell frequency correlated with tumor size (r = 0.742; P = 0.017). FoxP3+ regulatory T cell (Treg) frequency correlated with lymph node metastases (r = 0.858; P = 0.002), and CD8 to Treg ratio correlated inversely with tumor size (r = −0.804; P = 0.007). Conclusions: TAL and high Treg frequency in primary thyroid tumors correlates with more aggressive disease. Future prospective studies may identify Treg frequency as a predictive factor in PTC, and the suppressive effects of Treg should be considered in the design of immune-based therapies.

The phenomenon of field cancerization has been previously postulated and observed in various cancers, including those of the lung. We have recently demonstrated that normal-appearing airway cells carry expression profiles that are often characteristic of the adjacent tumor. A better understanding of the molecular mechanisms that drive these field changes may provide important biological insights into lung cancer pathogenesis. Loss-of-heterozygosity (LOH) and other forms of acquired chromosomal alterations (allelic imbalance; AI) have an established and profound role in oncogenesis. However, the relationship between AI and field cancerization has not been studied comprehensively across the genome. Here we address this void by interrogating a rich collection of normal-appearing airways from non-small cell lung cancer (NSCLC) patients. We applied Illumina 1M SNP arrays to characterize whole genome copy number alterations in 435 samples from 45 early-stage NSCLC patients [31 adenocarcinomas (ADCs), 14 squamous cell carcinomas (SCCs)]. Each patient set comprised samples from the primary tumor and adjacent normal-appearing airways paired with blood cells and/or uninvolved normal lung tissue. A subset of these included brushings from ipsilateral large airways (mainstem bronchi) and from the nasal cavities as well as multi-region tumor biopsies for intra-tumoral analysis (on 22, 27, and 20 patients, respectively). To characterize the field in normal-appearing airways at a genome-wide scale, we applied a haplotype-based computational program, hapLOH, to profile AI events (loss, gain, copy neutral LOH) in a paired mode contrasting signals in the blood or normal lung. We detected 198 AI events in normal-appearing airways of 24 of 45 patients; 92% of these events were represented in the paired tumor. Of the 24 patients, 23 had events in the adjacent airway, 3 had events in the large airway, and no events were observed in nasal brushings, indicating a pronounced AI field gradient. We detected AI in the airways of approximately 43% of ADCs regardless of smoking status (3 of 7 smokers, 10 of 24 non-smokers), and 79% (11 of 14) of SCCs, clearly indicating squamous histology as a greater predictor of observing a genomic field effect (P < 0.03). The most frequently observed airway alterations were in 9p and 9q, affecting 13 smoker patients; interestingly, these were not observed in the non-smokers. Finally, we note that AI events were present in the adjacent airways of 4/5 (80%) of patients with recurrence and only in 20/40 patients without recurrence, signaling a profound prognostic value in studying the field of cancerization in NSCLC. Although preliminary, our findings suggest that chromosomal aberrations are common in the normal-appearing airway field of cancerization and can potentially provide insights into the biology of lung cancer pathogenesis and progression. Citation Format: Yasminka Jakubek, Wenhua Lang, Selina Vattathil, Melinda Garcia, Lili Huang, Wei Lu, Chi-Wan Chow, Zachary Weber, Gareth E. Davies, Carmen Behrens, Neda Kalhor, Cesar Moran, Junya Fujimoto, Reza J. Mehran, Jerry Fowler, Erik A. Ehli, Ignacio I. Wistuba, Paul Scheet, Humam Kadara. The landscape of DNA allelic imbalance in the normal-appearing airway field of cancerization. [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 2993. doi:10.1158/1538-7445.AM2015-2993