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Context A histologic classification of lung cancer subtypes is essential in guiding therapeutic management. Objective To complement morphology-based classification of lung tumors, a previously developed lung subtyping panel (LSP) of 57 genes was tested using multiple public fresh-frozen gene-expression data sets and a prospectively collected set of formalin-fixed, paraffin-embedded lung tumor samples. Design The LSP gene-expression signature was evaluated in multiple lung cancer gene-expression data sets totaling 2177 patients collected from 4 platforms: Illumina RNAseq (San Diego, California), Agilent (Santa Clara, California) and Affymetrix (Santa Clara) microarrays, and quantitative reverse transcription–polymerase chain reaction. Gene centroids were calculated for each of 3 genomic-defined subtypes: adenocarcinoma, squamous cell carcinoma, and neuroendocrine, the latter of which encompassed both small cell carcinoma and carcinoid. Classification by LSP into 3 subtypes was evaluated in both fresh-frozen and formalin-fixed, paraffin-embedded tumor samples, and agreement with the original morphology-based diagnosis was determined. Results The LSP-based classifications demonstrated overall agreement with the original clinical diagnosis ranging from 78% (251 of 322) to 91% (492 of 538 and 869 of 951) in the fresh-frozen public data sets and 84% (65 of 77) in the formalin-fixed, paraffin-embedded data set. The LSP performance was independent of tissue-preservation method and gene-expression platform. Secondary, blinded pathology review of formalin-fixed, paraffin-embedded samples demonstrated concordance of 82% (63 of 77) with the original morphology diagnosis. Conclusions The LSP gene-expression signature is a reproducible and objective method for classifying lung tumors and demonstrates good concordance with morphology-based classification across multiple data sets. The LSP panel can supplement morphologic assessment of lung cancers, particularly when classification by standard methods is challenging.

Introduction Molecular subtyping of lung adenocarcinoma (AD) and lung squamous cell carcinoma (SCC) reveal biologically diverse tumors that vary in their genomic and clinical attributes. Methods Published immune cell signatures and several lung AD and SCC gene expression data sets, including The Cancer Genome Atlas, were used to examine immune response in relation to AD and SCC expression subtypes. Expression of immune cell populations and other immune related genes, including CD274 molecule gene ( CD274 ) (programmed death ligand 1), was investigated in the tumor microenvironment relative to the expression subtypes of the AD (terminal respiratory unit, proximal proliferative, and proximal inflammatory) and SCC (primitive, classical, secretory, and basal) subtypes. Results Lung AD and SCC expression subtypes demonstrated significant differences in tumor immune landscape. The proximal proliferative subtype of AD demonstrated low immune cell expression among ADs whereas the secretory subtype showed elevated immune cell expression among SCCs. Tumor expression subtype was a better predictor of immune cell expression than CD274 (programmed death ligand 1) in SCC tumors but was a comparable predictor in AD tumors. Nonsilent mutation burden was not correlated with immune cell expression across subtypes; however, major histocompatibility complex class II gene expression was highly correlated with immune cell expression. Increased immune and major histocompatibility complex II gene expression was associated with improved survival in the terminal respiratory unit and proximal inflammatory subtypes of AD and in the primitive subtype of SCC. Conclusions Molecular expression subtypes of lung AD and SCC demonstrate key and reproducible differences in immune host response. Evaluation of tumor expression subtypes as potential biomarkers for immunotherapy should be investigated.

4558 Background: Objective: To examine in a cohort of anti-PD-(L)1 immune checkpoint inhibitors (ICP) treated urothelial cancer patients a strategy combining treatment outcomes with molecular alterations, pathways, and immune/tumor microenvironment features to determine potential responder and rapid-progression signatures. Methods: De-identified clinical history and treatment outcomes were collected on 109 MIBC patients treated with ICP agents. Archived FFPE samples from these patients were obtained and processed for mRNAseq, exome-seq, tumor mutation burden (TMB), microsatellite instability (MSI) and mutation panel testing. Comprehensive tumor/immune profiling is being analyzed in the context of ICP treatments and RECIST 1.1 outcomes. A 60 gene MIBC 4-typer expression subtyper and other response associated predictors are used to stratify and identify positive/negative ICP response indicators. Results: 109 patients were identified (median age 75, 64% male, 78% white, 17% black). 74% of patients had received prior platinum-based chemotherapy, and 12% had received 2 or more prior lines of therapy. At initiation of ICP, 28% of patients had hemoglobin < 10, 30% had liver metastases, and 59% had ECOG performance status > 0. Mutation analysis of the first 66 patients showed TP53 (n = 34, 52%), FGFR (n = 17, 26%), CDKN2A (n = 13, 20%) and RB1 (n = 12, 18%) as the top alterations. No patients (0/8) with known pathogenic mutations in FGFR3 (S249C and TACC3-fusion) responded to ICP. Of patients with T2 staging prior to ICP (37/66), overall survival was markedly shorter (2.7 years) in those possessing FGFR3 mutations (n = 6/37) compared to that for FGFR3 WT patients (5.7 years, n = 31/37; p = 0.045). Further analyses of molecular features relative to treatment outcomes are ongoing to characterize response signatures. Conclusions: Our preliminary cohort of patients with pathogenic FGFR3 alterations showed 0% favorable response to ICP. We are expanding on this observation with further comprehensive molecular analyses and retrospective treatments/outcomes data. We anticipate identifying expression signatures that reflect ICP patient responder/non-responder signatures that may aid in future therapy decisions.

4538Background: Gene expression subtypes provide valuable insight into tumor biology and potential therapeutic response. Differential expression of immune infiltrating cells and biomarkers, tumor m...

Background: Gene expression-based subtyping in lung adenocarcinoma (AD) and lung squamous cell carcinoma (SQ) classifies AD and SQ tumors into distinct subtypes with variable expression of underlying biology including DNA damage response genes. These subtypes are linked to differences in chemotherapy sensitivity, and may impact response to therapeutics like PARP inhibitors. Methods: Using The Cancer Genome Atlas (TCGA) lung cancer gene expression datasets (AD n=515 and SQ n=501), AD subtypes (Terminal Respiratory Unit (TRU), Proximal Proliferative (PP), and Proximal Inflammatory (PI)) and SQ subtypes (Primitive, Classical, Secretory, and Basal) were defined using gene expression based centroid predictors. Association between AD and SQ expression subtypes and 3 published BRCAness/PARP inhibitor response signatures developed in ovarian and/or breast cancer (Konstantinopoulos et al., PMID 20547991; Daemen et al., PMID 22875744; McGrail et al., PMID 28649435) was examined using linear regression. Association between subtypes and expression of 15 recognized homologous recombination (HR) related genes (ATM, ATR, BRCA1, BRCA2, BRIP1, CDK12, CHEK1, CHEK2, FANCA, FANCI, FANCD2, MRE11A, RAD51C, RAD51L1, PTEN) was also examined using linear regression, and association tests included adjustment for the 3 BRCAness/PARP inhibitor response signatures and proliferation score. Results: AD and SQ subtypes showed strong association with the 3 BRCAness/PARP inhibitor response signatures (F-test p-values 7.7e-05, 5.9e-13, 9.4e-33 in AD and 1.9e-05, 9.0e-13, 2.7e-19 in SQ). AD and SQ subtypes showed strong association with 15 HR genes (max and median F-test p-values were 8.5e-04 and 7.5e-25 in AD, and 7.3e-04 and 1.4e-12 in SQ). The TRU subtype in AD showed low expression relative to the other AD subtypes for a majority of the HR genes, including BRCA1. In SQ, the same was true for the basal and secretory subtypes. Simultaneous adjustment for the 3 BRCAness/PARP inhibitor response signatures, as well as for proliferation, reduced association strength between subtype and HR gene expression in AD and less so in SQ. In AD, association between subtype and gene expression remained significant for 4 HR genes (using Bonferroni correction for 15 tests), including CHECK2, FANCI, BRIP1, and RAD51L1. In SQ, association between subtype and gene expression remained significant for all HR genes except CHEK1 and FANCA, (median and min Bonferroni-adjusted p-value 2.9e-04 and 2.6e-21). Conclusions: Intrinsic biologic subtypes of lung AD and SQ are associated with published BRCAness/PARP inhibitor response signatures and reveal differential expression of several HR-related genes. Evaluation of these subtypes, particularly in SQ, as potential biomarkers of PARP inhibitor sensitivity should be investigated. Citation Format: Gregory Mayhew, Chuck Perou, D Neil Hayes, Myla Lai-Goldman, Hawazin Faruki. Differences in BRCAness/PARP inhibitor response signatures and homologous recombination gene expression across lung adenocarcinoma and squamous cell carcinoma gene expression subtypes [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A037.

A pharmacogenetic marker for abacavir hypersensitivity is rapidly being incorporated into routine medical practice following demonstration of strong clinical utility in pivotal clinical studies. As one of the few pharmacogenetic markers that have crossed from research tools to clinical adoption and utilization, the abacavir hypersensitivity pharmacogenetic marker provides a great model for demonstration of factors that are critical to successful pharmacogenetic test adoption. Several examples of novel diagnostic test implementation are reviewed with focus on factors that are critical to translation into clinical practice. Other pharmacogenetic markers that have not yet been integrated into routine clinical care are discussed and reasons for their lack of acceptance are suggested.

The assimilation of new genomic-based diagnostic capabilities into routine medical practice will undoubtedly have a profound impact on patient management decisions. However, the challenge of translating newly discovered biomarkers into widely utilized medical diagnostic applications remains. This chapter will discuss many of the technical, regulatory, and financial challenges to full adoption of genomic tests in the clinic, and will delineate some of the key drivers of test acceptance along a continuum from early discovery to full utilization.

Telepathology usage in the past has typically been a qualitative procedure rather than a quantitative measurement. DNA ploidy using image analysis has been favorably compared to DNA ploidy analysis by flow cytometry in numerous publications. A step from DNA ploidy analysis using conventional image analysis to DNA ploidy analysis using stored images allows DNA ploidy analysis by image cytometry to become a powerful tool in telepathology. Remote DNA ploidy analysis using stored images has an impact on the field of pathology, as not every hospital or laboratory can afford to perform this type of specialized testing. However, images have large data files and require lengthy transmission times over communication systems to other computers. Joint Photographer Experts Group (JPEG) compression is a computer algorithm that allows the file size of an image to be reduced in order to decrease transmission times to another computer. A study was initiated to investigate the effects of JPEG compression on images of Feulgen stained breast tumor touch preps and the resulting DNA ploidy histograms.

Although several morphological and molecular genetic studies have implicated various grades of pancreatic duct hyperplasia as precursor lesions to infiltrating pancreatic adenocarcinoma, the identity of preinvasive pancreatic neoplasms remains controversial. In the present study, the authors examined the expression of the epidermal growth factor receptor homologue, HER-2/ neu (c-erbB-2), in pancreatic duct lesions adjacent to infiltrating pancreas cancers in a series of 19 cases of pancreatic duct adenocarcinoma. HER-2/ neu expression was examined because it has been identified in a proportion of infiltrating pancreas cancers and because it may provide early neoplasms with a growth advantage over adjacent nonneoplastic epithelium. In normal pancreatic ducts and ductules, HER-2/ neu expression was absent in all but one case. By contrast, HER-2/ neu was expressed in 82% ( P = .008 vs normal) of ducts with flat mucinous hyperplasia, 86% ( P = .03 vs normal) of ducts with papillary mucinous hyperplasia without atypia, 92% ( P = .001 vs normal) of ducts with atypical papillary mucinous hyperplasia, and all specimens with carcinoma in situ. HER-2/ neu expression was observed in 69% ( P = .002 vs normal) of the moderately differentiated infiltrating carcinomas and none of the poorly differentiated infiltrating carcinomas. These data establish HER-2/ neu as a potential mediator of growth factor-related signal transduction in pancreatic duct lesions, and provide additional support for the hypothesis that lesions formerly regarded as various grades of hyperplasia instead may represent intraepithelial neoplasms with the potential for subsequent invasion and metastasis.

3077Background: Gene expression based subtyping in lung Adenocarcinoma (AD) and lung Squamous Cell Carcinoma (SQ) classifies AD and SQ tumors into distinct subtypes with variable biologic and clini...

HLA-B*5701 testing to provide risk stratification for abacavir hypersensitivity has the potential to reduce incidence of hypersensitivity reactions in susceptible individuals. Early experience with clinical HLA-B*5701 testing of the first 100 specimens, from a large clinical reference laboratory in the United States, is presented. Patient samples were tested using a two-step approach. The first step allowed rapid identification of most HLA-B*5701-negative samples in a high throughput mode. The second step involved resolution of putative positives by DNA sequencing to identify B*5701 specifically as well as other B57 subtypes. Test reporting included a phone call from a genetic counselor to obtain the ethnic background and indication for testing and to provide a patient-specific interpretation. The patients population was comprised of Caucasians, 84%; Hispanics, 13%; and African Americans, 3%. Among the 100 samples tested, 92% were HLA-B*5701-negative and 8% were positive for the HLA-B*5701 allele. All HLA-B*5701 allele positives were identified in Caucasian patients. Where the indication for testing was obtainable (57 patients), pre-abacavir therapy screening was the indication 67% of the time. Clarification of previous suspected history of hypersensitivity was the indication 33% of the time. Among samples tested to help clarify a previous history of hypersensitivity, 16/19 or 84% did not carry the HLA-B*5701 allele whereas 3/19 (16%) were carriers of the HLA-B*5701 allele. Early utilization of HLA-B*5701 testing in community practice was not always consistent with the clinical indications for testing. Post-test communication assisted in providing physician education and interpretation of patient-specific results.

Background Stool-based DNA screening for colorectal cancer (CRC) was recently made available for use in daily clinical practice (PreGen-PlusTM). The main objectives of this study were to examine patients' screening experiences with stool DNA testing in routine clinical practice and the results of diagnostic colonoscopy in patients with an antecedent abnormal stool DNA test. Patients and Methods Patients undergoing stool-based DNA testing were asked to complete and return via mail an anonymous 10-item questionnaire inquiring about their test-related experiences. Colonoscopy findings for all abnormal stool-based DNA tests were ascertained via a telephone survey of the ordering primary care clinicians' offices. Results Patient survey responses were collected between August 2003 and July 2005 and reflect an 18% (1211 of 6730) response rate. The majority reported that the specimen collection process was very easy/easy to perform (87%), that they were very likely/likely to use the test again (91%), and that they had never been screened for CRC previously by any method (52%). Tests were ordered predominantly by the patient's primary care clinician (90%), including obstetrician/gynecologist providers. Colonoscopy findings from 69 of 159 patients with an antecedent abnormal stool DNA test screened with PreGen-PlusTM between August 2003 and July 2004 were available for review. An abnormal stool DNA test correlated with a colonoscopically demonstrable abnormality in 49% of cases (34 of 69). Abnormal findings, including CRC in 3 patients (4%; 1 with Dukes A and 2 with Dukes B disease), single or multiple adenomatous polyps in 23 patients (33%), hyperplastic polyps in 3 patients (4%), and colitis in 5 patients (7%). Colonoscopy was reported as negative in 51% of patients (35 of 69), including 2 cases (3%) with an altered BAT-26 microsatellite caused by a normal polymorphism. Conclusion Stool DNA testing provides an acceptable noninvasive alternative for CRC screening that can identify early-stage CRCs and adenomatous polyps in routine clinical practice. Ongoing and broader surveys are indicated to support these early findings.

7566 Background: Lung cancer subtypes are described by morphological and molecular characteristics. Differentiation between various subtypes is important for guiding patient management. Variability in morphology, limited tissue, and the need for performing a growing number of genomic tests pose challenges to the current diagnostic standard. Methods: The Lung Subtype Panel (LSP), a previously published 57 gene expression panel for prediction of lung tumor morphologic class (Wilkerson et al.JMD 2013), was investigated using 2,168 lung cancer samples from multiple publically available data sets (TCGA, NCI, UNC, Duke, Expo, Seoul, Tokyo, and France). Data sets with both gene expression data and morphologic classification were selected. Three platforms for gene expression were represented: Affymetrix U133+2 (n=883), Agilent 44K (n=334), and Illumina RNA-seq (n=951). A centroid predictor was used to assign an expression-defined histologic class. Predicted results were compared with tumor morphologic classificat...

7576 Background: Recommendations exist to aid the clinically relevant non-small cell lung cancer (NSCLC) classification of squamous (SQ) versus adenocarcinoma (AD), including immunohistochemistry (IHC) for TTF1 and p63. We hypothesize that PCR of RNA gene panels may improve the diagnostic accuracy over IHC. Methods: A multi-institutional cohort of NSCLC patients was abstracted for use of IHC in clinical practice. RNA was isolated from routine paraffin sections and a 54-gene Histology by gene Expression Predictor (HEP) was implemented to distinguish SQ, AD, and other NSCLC variants. IHC for TTF1 and p63 was obtained on a subset of patients in a standardized manner. To compare the reproducibility of PCR diagnostics to morphologic diagnosis, a subset of samples was processed as matched pairs from the same tumor, in blinded manner. Results: 493 clinical samples were analyzed from 419 patients. Pathologists obtained any IHC in 22% of the cases, with TTF1 or p63 in

The authors tested 15 immune serum globulin pharmaceutical preparations for antibody reactivity to human T cell lymphotrophic virus type III (HTLV-III) by the Abbott immunoenzymometric assay (IEMA). Although no evidence of HTLV-III infectivity has appeared after injection of similar preparations into humans, the authors found all samples IEMA reactive. Upon dilution, the authors demonstrated parallel decreases of antibody reactivity among two samples of gamma globulin, the Abbott-positive control, and a markedly reactive patient specimen. Gamma globulin isolated from sera of six animal species was nonreactive in the Abbott assay. All samples were nonreactive with the H-9 cell line antigen. Antibody reactivity to HTLV-III was confirmed in 13 of 15 gamma globulin samples when tested by the Electro-Nucleonics IEMA, and 14 samples contained at least the p24 band on Western blot analysis. Although false positivity occurs in IEMA assays possibly because of elevated protein concentrations and nonspecific binding, the authors, results show that in most circumstances immune serum globulin preparations tested do contain true reactivity to HTLV-III.