Travis Sullivan

Profiling microRNA from nephrectomy and biopsy specimens: predictors of progression and survival in clear cell renal cell carcinoma

To identify microRNA (miRNA) characteristic of metastatic clear cell renal cell carcinoma (ccRCC) and those indicative of cancer‐specific survival (CSS) in nephrectomy and biopsy specimens. We also sought to determine if a miRNA panel could differentiate benign from ccRCC tissue.

MicroRNA Expression Profile Identifies High Grade, Non-Muscle-Invasive Bladder Tumors at Elevated Risk to Progress to an Invasive Phenotype

The objective of this study was to identify a panel of microRNAs (miRNAs) differentially expressed in high-grade non-muscle invasive (NMI; TaG3–T1G3) urothelial carcinoma that progress to muscle-invasive disease compared to those that remain non-muscle invasive, whether recurrence happens or not. Eighty-nine high-grade NMI urothelial carcinoma lesions were identified and total RNA was extracted from paraffin-embedded tissue. Patients were categorized as either having a non-muscle invasive lesion with no evidence of progression over a 3-year period or as having a similar lesion showing progression to muscle invasion over the same period. In addition, comparison of miRNA expression levels between patients with and without prior intravesical therapy was performed. Total RNA was pooled for microarray analysis in each group (non-progressors and progressors), and qRT-PCR of individual samples validated differential expression between non-progressive and progressive lesions. MiR-32-5p, -224-5p, and -412-3p were associated with cancer-specific survival. Downregulation of miR-203a-3p and miR-205-5p were significantly linked to progression in non-muscle invasive bladder tumors. These miRNAs include those implicated in epithelial mesenchymal transition, previously identified as members of a panel characterizing transition from the non-invasive to invasive phenotype in bladder tumors. Furthermore, we were able to identify specific miRNAs that are linked to postoperative outcome in patients with high grade NMI urothelial carcinoma of the bladder (UCB) that progressed to muscle-invasive (MI) disease.

MicroRNA expression profiles in upper tract urothelial carcinoma differentiate tumor grade, stage and survival: implications for clinical decision-making

OBJECTIVE To evaluate microRNA (miRNA) biomarkers for upper tract urothelial carcinoma (UTUC) to improve risk stratification. METHODS miRNA was isolated from 157 radical nephroureterectomy specimens from 2 institutions. The relative expression of miRNA was examined for high grade vs low grade tumors as well as muscle invasive vs nonmuscle invasive tumors. Recurrence free survival (RFS) and overall survival (OS) were also stratified using relative expression of specific miRNA. RESULTS The optimized model to identify high grade UTUC included miR-29b-2-5p, miR-18a-5p, miR-223-3p, and miR-199a-5p, generating a sensitivity of 83%, specificity of 85%, and generated a receiver operating characteristic (ROC) curve with area-under-the-curve of 0.86. Similarly, the model classifier for predicting ≥pT2 disease incorporated miR-10b-5p, miR-26a-5p-5p, miR-31-5p, and miR-146b-5p, producing a sensitivity of 64%, specificity of 96%, and area-under-the-curve of 0.90. RFS was best reflected by a combination of miR-10a-5p, miR-30c-5p, and miR-10b-5p, while OS was best predicted by miR-10a-5p, miR-199a-5p, miR-30c-5p, and miR-10b-5p. CONCLUSION High-grade vs low-grade as well as muscle invasive vs nonmuscle invasive UTUC can be reliable distinguished with unique miRNA signatures. Furthermore, differential expression of UTUC miRNA produces robust classifiers for predicting RFS and OS that may help identify patients who would most benefit from adjuvant therapies.

Abstract A15: Nasal gene expression for the diagnostic evaluation of indeterminate pulmonary nodules within a screening population

Introduction: Lung cancer remains the leading cause of cancer-related death due to the limited ability to detect the disease at an early and potentially curable stage. CT lung screening (CTLS) of high-risk patients improves mortality. However, the high proportion of false-positive tests, the majority of which are indeterminate pulmonary nodules (IPNs), has limited widespread adoption of this life-saving screening modality. Since screen-detected IPNs contribute to the overall cost of screening and potential morbidity due to the need for further diagnostic testing, there is an urgent need for accurate tools to stratify patients with screen-detected nodules for routine follow-up or additional intervention. Prior work from our group has identified alterations in nasal epithelial cell gene expression in individuals undergoing bronchoscopy, which are associated with lung cancer. In this study, we are evaluating the ability of nasal epithelial gene expression to identify patients with lung cancer among CTLS patients with IPNs. Methods: Nasal epithelial brushings were collected from a screening cohort at Lahey Hospital & Medical Center (LHMC) with CTLS IPNs (6-20mm; n=38) that had been followed prospectively for up to 2 years (19 lung cancers; 19 benign disease). We performed total RNA sequencing on the nasal samples using the Illumina TruSeq Stranded Total RNA Sample Preparation kit. Reads were aligned to the human assembly Genome Reference Consortium Human Build 37 to identify a gene signature associated with cancer status. To evaluate the signature in an independent IPN cohort, nasal epithelial brushings were collected from current and former smokers (n=67; age>45, pack-year>20) undergoing diagnostic workup for IPNs (7-30 mm) at military and VA hospitals within the DECAMP consortium. Subjects were followed prospectively for up to two years after sample collection until a final diagnosis of lung cancer (n=38) or benign disease (n=29) was made. Results: Using a generalized linear model correcting for smoking status, we identified 39 differentially expressed genes (DEGs, FDR-q Conclusion: Our findings suggest that there are nasal gene expression alterations associated with lung cancer diagnosis among CTLS patients with indeterminate pulmonary nodules. These alterations may be leveraged to develop a nasal biomarker for the early detection of lung cancer in high-risk smokers. Citation Format: Katrina Steiling, Jiarui Zhang, Jacob Sands, Travis Sullivan, Ehab Billatos, Elizabeth Moses, Gang Liu, Carla Lamb, Brady McKee, Marc Lenburg, Avrum Spira, Kimberly Rieger-Christ. Nasal gene expression for the diagnostic evaluation of indeterminate pulmonary nodules within a screening population [abstract]. In: Proceedings of the Fifth AACR-IASLC International Joint Conference: Lung Cancer Translational Science from the Bench to the Clinic; Jan 8-11, 2018; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(17_Suppl):Abstract nr A15.

From transcriptional noise to modulator of the TGFB1 pathway, a player in the development of chemoresistance: lncRNA-LET

In order to win the battle against cancer, two hurdles need to be overcome; the development of drug resistance and the spread of cancer through metastasis.

MP88-15 DISTINCT EXOSOMAL MIRNA PROFILES IN CHEMORESISTANT BLADDER CARCINOMA CELL LINES

and pelvis remains the main site of local recurrence. We sought to investigate whether aggressiveness of bladder cancer correlated with washings in the pelvis & pneumo-peritoneum at different stages of robot-assisted radical cystectomy (RARC). METHODS: 20 patients who underwent RARC were prospectively enrolled in the study. 6 samples were collected from each patient: Intra-vesical washing prior to RARC (BW), followed by a series of 3 pelvic irrigations; before RARC (Wash 1), after RARC (Wash 2) and after pelvic lymph node dissection (PLND) (Wash 3). Leftover suction fluid from the whole procedure was also collected (Wash 4). A specialized filter from surgical smoke evacuation devicewas used to trap any cells circulating in the pneumoperitoneum. Each samplewas examined for cytology and the presence of bladder cancer-related markers (CDK1, HOXA13, MDK, IGFBP3). Results were correlated with clinical outcomes. RESULTS: 18 patients were included in the study (2 excluded for concomitant malignancies). MDK had the highest detection rate in the study. CDK1 had the lowest detection rate among the four markers and was only detected in 1 intra-vesical wash (pT1). There was no difference in the detection rate of the mRNAmarkers between muscle invasive and non-invasive tumors. However, mRNA in the pelvic irrigation and suction fluids were only detected in invasive (78%) and metastatic (100%) stages. Cytology results showed atypical cells in 4 patients (1 Wash 3, 3 Wash 4). After a median follow-up of 7 months, 2 patients developed distant recurrences (ypT0/N1, pT4a/N2) and 1 patient had pelvic recurrence (T4b/N0). No transmission of tumor cells was seen in the pneumo-peritoneum insufflation CO2 used during RARC. CONCLUSIONS: This simple novel methodology was able to provide valuable information regarding possible pelvic dissemination. Patients with more advanced disease after RARC may have higher odds of bladder cancer dissemination in the pelvis during RARC.

A live cell microfluidics device utilizing phenotypic biomarkers for prostate cancer.

338 Background: A novel tissue based biomarker panel is introduced to objectively assess disease aggressiveness and invasive potential of Prostate Cancer (CaP). The biomarker diagnostic platform incorporates both molecular and phenotypic data that may allow an improved understanding of local growth and metastatic potential. The tissue based diagnostic incorporates matrix biology, phenotypic biomarkers, microfluidics, and machine vision. This technology presents the opportunity to culture samples, and both determine and automate biomarker measurements from machine vision algorithm analysis. Data are presented towards clinical validation, the ability to risk stratify, and prediction of local aggressiveness and metastasis. Methods: Conditions were optimized for reliably culturing primary cancer cells in vitro by simulating in vivo conditions on an extracellular matrix formulation. A mcirofluidics device was used to culture live tumor samples ex vivo enabling automated imaging of the label free and label-base...

MP1-19 A NOVEL LIVE CELL MICROFLUIDIC DIAGNOSTIC USING PHENOTYPIC BIOMARKERS WITH OBJECTIVE ALGORITHMIC ANALYSIS FOR PROSTATE CANCER RISK STRATIFICATION.

PTEN IHC was 62% sensitive for detection of hemizygous PTEN deletion by FISH, with homogeneous or heterogeneous PTEN protein loss in 30/48 tumors with hemizygous deletion. Other inactivating mutations of PTEN (not detectable by FISH) may lead to PTEN protein loss in tumors with normal or hemizygous PTEN copy number. CONCLUSIONS: Our validated and automated PTEN immunostaining protocol is a simple and relatively inexpensive test that is 91% specific and 98% sensitive for detection of homozygous PTEN gene deletions in a large multi-center study of prostate cancer.

MP61-07 SERUM MICRORNA ANALYSIS: A MINIMALLY INVASIVE ASSAY CORRELATED WITH UPGRADING IN PATIENTS WITH LOW-RISK PROSTATE CANCER

INTRODUCTION AND OBJECTIVES: Pathologic upgrading from biopsy to prostatectomy occurs in approximately 30% of prostate cancer cases. PSA, a nonspecific protein, has previously been the primary serum marker for prostate cancer. Markers that more accurately assess risk of aggressiveprostate cancer at the timeof screening are critical for the future management of this disease. The aim of this study was to identify a panel of microRNA (miRNA) from serum that could differentiate patients with low-risk (Gleason 6) prostate cancer on TRUS biopsy specimenswho were either the same grade or upgraded at the time of prostatectomy. METHODS: Total RNA was isolated from serum of patients who had Gleason Sum (GS) 6 prostate cancer at the time of TRUS guided prostate biopsy. These patients were divided into groups with GS 6 that remained GS 6 (same grade) at prostatectomy and those who were upgraded to GS7 (upgrade) at the time of prostatectomy. For the discovery phase, sample pools from each group were profiled via miRNA PCR array (Exiqon). Validation of miRNA expression levels was performed on 29 same and 31 upgrade samples by qRT-PCR. RESULTS: Array analysis of 751miRNA identified34miRNAwith 2-fold differential expression between patients who had Gleason upgrading between biopsies and prostatectomy compared to patients who were same grade. Of these, 20 miRNA were down-regulated and 14 were up-regulated in theupgradedgroup.Additionally, 3miRNAwereexpressed in the upgrade groupwhich were not detected in the same grade group. 17 miRNA were further validated by qRT-PCR on individual samples. miR425-5pandmiR-146-5pwere found tobesignificantlydifferent between the same grade and upgraded groups. ROC curve of logistic regression analysis of theses two showed an area under the curve of 0.691. CONCLUSIONS: Serum samples demonstrated different miRNA expression levels between samples that were same grade or upgraded from Gleason 6 at prostatectomy. This minimally invasive assay could provide an adjunct to PSA and prostate biopsies to better counsel patients on management of low-risk prostate cancer and monitor patients on active surveillance.

Abstract 4347: A novel live cell diagnostic platform measuring phenotypic biomarkers using objective algorithmic analysis enables further risk stratification for intermediate-risk prostate cancer patients

Due to the inconsistencies of existing molecular, genomic, and pathophysiologic markers for patient risk stratification, effective prostate cancer diagnostics and treatment remains a challenge in clinical practice. Therefore, the development of a diagnostic platform that differentiates cancer patients who have clinically significant disease from those who have a low risk of progression is an important area of interest. In this study, we tested a diagnostic platform that combines a scalable microfluidic device, an automated live cell assay, and objective machine vision algorithms to measure phenotypic biomarkers [defined here as functional biophysical and molecular biomarkers], which evaluate both local growth and metastatic potential of prostate cancer. An analytical validation study was performed on fresh prostate cancer samples (n = 100) obtained at the time of radical prostatectomy (RP). The diagnostic platform enables: 1) growth of patient cells ex vivo on extra cellular matrix formulations supporting adhesion/survival for 72 hours 2) high-throughput imaging of multiple phenotypic biomarkers such as morphology, cytoskeleton dynamics, and protein subcellular localization & modification states and 3) objective quantification of biomarkers via machine vision analysis. Patient samples were imaged over a three hour period capturing live-cell biophysical biomarkers. After three hours cells were fixed and stained for molecular biomarkers. Machine vision technology was then utilized to analyze phenotypic biomarkers to yield specific metrics that quantified local tumor growth (Oncogenic Potential-OPs) and invasive potential of the tumor to other tissues (Metastatic Potential- MPs) that correlated with RP specimen pathologic findings. Analysis of quantified phenotypic biomarkers distinguished normal cells from cancer cells. The OP and MP metrics demonstrated statistical significance in distinguishing Gleason 6 (low-risk) from Gleason 7 (intermediate-risk) prostate cancer with 80% sensitivity and 80% specificity and concordance with relevant RP pathology findings. Specifically, OP and MP derived from defined phenotypic biomarker metrics, demonstrated the ability to differentiate Gleason 6 and 7 scores and correlated with, 1) seminal vesicle invasion, 2) positive RP surgical margins, 3) vascular invasion, and 4) lymph node involvement. This novel functional-live-cell diagnostic platform allows for the measurement of a biomarker panel that further stratifies patients to improve prostate cancer treatment, clinical decision-making, further risk stratification of intermediate prostate cancer populations, and potentially predict actionable pathological findings leading to improved treatment outcomes for prostate cancer patients. Citation Format: Michael S. Manak, Wendell R. Su, Andrew Min, Brad J. Hogan, Matthew J. Whitfield, Jonathan S. Varsanik, Delaney Berger, Mani Foroohar, Kimberly M. Rieger-Christ, Travis B. Sullivan, Naveen Kella, Ray Hernandez, Vladimir Mouraviev, Kevin B. Knopf, Hani H. Rashid, David M. Albala, Grannum R. Sant, Ashok C. Chander. A novel live cell diagnostic platform measuring phenotypic biomarkers using objective algorithmic analysis enables further risk stratification for intermediate-risk prostate cancer patients. [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 4347. doi:10.1158/1538-7445.AM2015-4347