Search

Feature-region combination AUCs and benchmarking Sheet 1: Log2 fold-change, p-value, and q-value between ARPC and NEPC lines for central mean coverage in all queried (338) TFs (two tailed Mann-Whitney U test, Benjamini-Hochberg adjusted). Sheet 2: 100-fold cross-validation AUCs for all region and feature combinations subset by the ‘AR10’ overlapping features (see Methods). Sheet 3: 100-fold cross-validation AUCs for all region and feature combinations subset by the ‘Phenotype 47-defining’ overlapping features. Sheet 4: 100-fold cross-validation AUCs for all region and feature combinations (global). Sheet 5: Predictions scores, tumor fraction, depth of coverage, and subtype for benchmarking admixtures. Sheet 6: AUCs for unsupervised prediction of admixture subtypes grouped by tumor fraction and depth. Sheet 7: NEPC:ARPC ratio, tumor fraction, and ARPC and NEPC fraction predictions for mixed phenotype admixtures using Keraon (see Methods).

Supplementary Figures S1-S18 and Supplementary Table legends.

Supplementary Tables

Purpose:Neuroendocrine prostate cancer (NEPC) is an aggressive form of castration-resistant prostate cancer (CRPC) for which effective therapies are lacking. We previously identified carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5) as a promising NEPC cell surface antigen. Here we investigated the scope of CEACAM5 expression in end-stage prostate cancer, the basis for CEACAM5 enrichment in NEPC, and the therapeutic potential of the CEACAM5 antibody–drug conjugate labetuzumab govitecan in prostate cancer.Experimental Design:The expression of CEACAM5 and other clinically relevant antigens was characterized by multiplex immunofluorescence of a tissue microarray comprising metastatic tumors from 34 lethal metastatic CRPC (mCRPC) cases. A genetically defined neuroendocrine transdifferentiation assay of prostate cancer was developed to evaluate mechanisms of CEACAM5 regulation in NEPC. The specificity and efficacy of labetuzumab govitecan was determined in CEACAM5+ prostate cancer cell lines and patient-derived xenografts models.Results:CEACAM5 expression was enriched in NEPC compared with other mCRPC subtypes and minimally overlapped with prostate-specific membrane antigen, prostate stem cell antigen, and trophoblast cell surface antigen 2 expression. We focused on a correlation between the expression of the pioneer transcription factor ASCL1 and CEACAM5 to determine that ASCL1 can drive neuroendocrine reprogramming of prostate cancer which is associated with increased chromatin accessibility of the CEACAM5 core promoter and CEACAM5 expression. Labetuzumab govitecan induced DNA damage in CEACAM5+ prostate cancer cell lines and marked antitumor responses in CEACAM5+ CRPC xenograft models including chemotherapy-resistant NEPC.Conclusions:Our findings provide insights into the scope and regulation of CEACAM5 expression in prostate cancer and strong support for clinical studies of labetuzumab govitecan for NEPC.

Supplementary Figures and Supplementary Materials and Methods

This formal comment is in response to “A quest for miRNA bio-marker: a track back approach from gingivo buccal cancer to two different types of precancers” written by De Sarkar and colleagues in 2014. The above-mentioned paper found seven miRNAs to be significantly deregulated in 18 gingivo-buccal cancer samples. However, they suspected more miRNAs to be deregulated based on their exploratory statistical analysis. To control the false discovery rate (FDR), the authors used the Benjamini Hochberg (BH) method, which does not leverage any available biological information on the miRNAs. In this work, we show that some specialized versions of the BH method, which can exploit positional information on the miRNAs, can lead to seven more discoveries with this data. Specifically, we group the closely located miRNAs, and use the group Benjamini Hochberg (GBH) methods (Hu et al., 2010), which reportedly have more statistical power than the BH method (Liu et al., 2019). The whole transcriptome analysis of Sing et al. (2017) and previous literature on the miRNAs suggest that most of the newly discovered miRNAs play a role in oncogenesis. In particular, the newly discovered miRNAs include hsa-miR-1 and hsa-miR-21-5p, whose cancer-related activities are well-established. Our findings indicate that incorporating the GBH method into suitable microarray studies may potentially enhance scientific discoveries via the exploitation of additional biological information.

Advanced prostate cancers comprise distinct phenotypes, but tumor classification remains clinically challenging. Here, we harnessed circulating tumor DNA (ctDNA) to study tumor phenotypes by ascertaining nucleosome positioning patterns associated with transcription regulation. We sequenced plasma ctDNA whole genomes from patient-derived xenografts representing a spectrum of androgen receptor active (ARPC) and neuroendocrine (NEPC) prostate cancers. Nucleosome patterns associated with transcriptional activity were reflected in ctDNA at regions of genes, promoters, histone modifications, transcription factor binding, and accessible chromatin. We identified the activity of key phenotype-defining transcriptional regulators from ctDNA, including AR, ASCL1, HOXB13, HNF4G, and GATA2. To distinguish NEPC and ARPC in patient plasma samples, we developed prediction models that achieved accuracies of 97% for dominant phenotypes and 87% for mixed clinical phenotypes. Although phenotype classification is typically assessed by IHC or transcriptome profiling from tumor biopsies, we demonstrate that ctDNA provides comparable results with diagnostic advantages for precision oncology. Significance: This study provides insights into the dynamics of nucleosome positioning and gene regulation associated with cancer phenotypes that can be ascertained from ctDNA. New methods for classification in phenotype mixtures extend the utility of ctDNA beyond assessments of somatic DNA alterations with important implications for molecular classification and precision oncology. This article is highlighted in the In This Issue feature, p. 517

Bipolar androgen therapy (BAT) results in rapid fluctuation of testosterone (T) between near-castrate and supraphysiological levels and has shown promise in metastatic castration-resistant prostate cancer (mCRPC). Its clinical effects may be mediated through induction of DNA damage, and preclinical studies suggest synergy with PARP inhibitors.This was a single-center, Phase II trial testing olaparib plus BAT (T cypionate/enanthate 400 mg every 28 days) with ongoing androgen deprivation. Planned recruitment was 30 subjects (equal proportions with/without homologous recombination repair [HRR] gene mutations) with mCRPC post abiraterone and/or enzalutamide. The primary objective was to determine PSAThirty-six patients enrolled and 6 discontinued prior to response assessment. In the ITT cohort, PSABAT plus olaparib is associated with high response rates and long PFS. Clinical benefit was observed regardless of HRR gene mutational status.

BACKGROUND Among men with metastatic prostate cancer, about 10% have germline alterations in DNA damage response genes. Most studies have examined BRCA2 alone or an aggregate of BRCA1/2 and ATM. Emerging data suggest that ATM mutations may have distinct biology and warrant individual evaluation. The objective of this study is to determine whether response to prostate cancer systemic therapies differs between men with germline mutations in ATM (gATM) and BRCA2 (gBRCA2). METHODS This is an international multicenter retrospective matched cohort study of men with prostate cancer harboring gATM or gBRCA2. PSA50 response (≥50% decline in prostate-specific antigen) was compared using Fisher's exact test. RESULTS AND LIMITATIONS The study included 45 gATM and 45 gBRCA2 patients, matched on stage and year of germline testing. Patients with gATM and gBRCA2 had similar age, Gleason grade, and PSA at diagnosis. We did not observe differences in PSA50 responses to abiraterone, enzalutamide, or docetaxel in metastatic castration resistant prostate cancer between the two groups; however, 0/7 with gATM and 12/14 with gBRCA2 achieved PSA50 response to PARPi (p

Introduction: Metastatic castration-resistant prostate cancer (mCRPC) is a heterogeneous disease which can be classified into clinically relevant subtypes based on the expression of transcription factors (TF), such as the androgen receptor (AR) and neuroendocrine markers. Neuroendocrine prostate cancer (NEPC), characterized by gain of stem-like and neuroendocrine features and lack of AR expression is a clinically aggressive variant. Due to the absence of adequate biomarkers, NEPC is usually detected at a very advanced stage. There is mounting evidence that molecular subtype changes seen in NEPC are enforced by widespread epigenetic alterations, in particular DNA methylation changes. In this study, we aim to devise a novel DNA methylation-based assay for molecular subtyping and disease monitoring from cell-free DNA (cfDNA). Methods: We analyzed genome wide methylation patterns in 60 prostate cancer patient-derived xenograft (PDX) and 133 mCRPC tumors using array- and sequencing-based assays. We integrated DNA methylation with TF cistrome data to determine the landscape of methylation alterations at key lineage TF binding sites (TFBS). A linear regression model was trained on low-pass Enzymatic Methyl-Seq (EM-seq) cfDNA data derived from PDXs to identify molecular subtype specific DNA methylation changes at these TFBS. The model performance was optimized with in silico admixture experiments. This model was then used to discern tumor molecular phenotypes from cfDNA in three independent cohorts of mCRPC patients using low-pass whole genome bisulfite sequencing and EM-seq. Results: We observed a strong association between TFBS methylation and TF expression. For lineage specific TFs such as AR and ASCL1, we identified core sets of TFBSs whose differential methylation allowed for accurate assay-independent molecular subtype classification in tumor tissues. Applying an optimized quantitative model to mCRPC patients who underwent comprehensive tissue sampling by rapid autopsy we observed perfect subtype prediction from both tissue samples and cfDNA (AUC=1). A similar analytical performance was observed in additional clinical mCRPC cohorts with cfDNA. Conclusions: We show that methylation patterns at TFBSs can determine TF activity and can be used to classify molecular subtypes from both tumor tissue and cfDNA. For prostate cancer, we demonstrate that this approach can accurately detect NEPC by cost-effective low-pass EM-seq. More broadly, this study provides a novel analysis framework for robustly assessing molecular tumor phenotypes in cfDNA with applications in solid and liquid tumor diagnostics. Citation Format: Mohamed Adil, Brian Hanratty, Pallabi Mustafi, Ilsa Coleman, Radhika Patel, Anna-Lisa Doebley, Robert Patton, Eden Cruikshank, Patricia Galipeau, Ruth Dumpit, Martine Roudier, Jin-Yih Low, Navonil De Sarkar, Robert Montgomery, Eva Corey, Colm Morrissey, Peter Nelson, Gavin Ha, Michael Haffner. Molecular phenotype classification of metastatic prostate cancer by cell-free DNA methylation analysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB298.

Background: With the adoption of potent AR signaling inhibitors, there has been a prominent rise in the frequency of treatment-resistant tumor phenotypes. Highly aggressive transdifferentiated tumors may emerge as AR low or AR null prostate cancers (PCa) with or without neuroendocrine signatures. An accurate determination of these epigenetically diverse phenotypic subtypes has direct clinical implications, including therapy choice. Current approaches for disease subtype determination often require tissue biopsies, limiting due to the high risk of comorbidity. For a temporally evolving cancer, a minimally invasive molecular assay is urgently needed to determine disease subtypes accurately to guide clinical decisions. Methods & Results: Cell-free DNA assay emerging as a nextgen tool for cancer early detection and real-time tumor evolution monitoring tool. Cell-free DNA primarily consists of DNA protected by nucleosomes and DNA binding elements and is shed into the bloodstream by cells that undergo apoptosis. We hypothesized that a nucleosome protection map and transcriptional regulation could be reliably determined from circulating tumor DNA (ctDNA) analysis. This analysis can accurately classify advanced prostate cancer into epigenetically divergent subclasses such as adenocarcinoma (ARPC) and neuroendocrine (NEPC). For developing robust phenotype-specific signatures, we performed whole-genome sequencing on plasma cfDNA from 30 PDX lines to achieve moderately deep coverage (>10X) of “pure” human ctDNA sequence data. We applied an analysis framework “Griffin” to determine transcription factor binding sites and transcription start site nucleosome protection signatures, predictive of specific transcription factor activity. We also generated H3K27ac, H3K27me1, and H3K27me3 histone profiles from resected PDX tumors and observed that ctDNA fragmentation and coverage patterns are consistent with histone modifications. Guided by differential nucleosome profiles, we derived a novel ctDNA fragment size variability signature that accurately predicted expression deregulation for phenotype-defining genes. These signatures helped us build an unsupervised statistical model that accurately classified PCa subtypes. We applied our model to 2 clinical cohorts and attained 96% accuracy for classifying patients with well-defined subtypes of advanced PCa, using low coverage sequencing. Conclusion: We have developed a ctDNA analysis method that can accurately classify epigenetically diverse subtypes of advanced prostate cancer, spanning AR active and neuroendocrine subtypes. This scalable and cost-effective approach is also suitable for longitudinal monitoring and prediction of treatment-emergent prostate cancer resulting from therapeutic resistance. Citation Format: Navonil De Sarkar. Native Cell-Free DNA sequence analysis reliably infers epigenetically diverse subtypes of prostate cancer. [abstract]. In: Proceedings of the AACR Special Conference: Precision Prevention, Early Detection, and Interception of Cancer; 2022 Nov 17-19; Austin, TX. Philadelphia (PA): AACR; Can Prev Res 2023;16(1 Suppl): Abstract nr P008.

Aim: The methylome associated with miRNA loci was investigated in oral cancer to explore tobacco specific methylation and potential biomarkers for patient survival. Methods: Methylome data was generated from 16 pairs of cancer-normal tissues by reduced representation bisulfite sequencing method. Differentially methylated regions were identified using the DMAP pipeline. In silico validation and Kaplan–Meier survival analyses were performed on The Cancer Genome Atlas data based on our miRNA methylome data. Results: A total of 4310 unique differentially methylated regions, mapping to 144 miRNA loci, were identified. Three distinct groups of miRNAs were differentially methylated in cancer tissues from smokers, chewers and mixed habitués. Hypermethylation of miR-503, miR-200a/b, miR-320b and miR-489 was associated with worse 5-year survival. Conclusion: Differential methylation patterns in miRNA loci are associated with poor survival underscoring their potential as predictive and prognostic biomarkers in oral cancer.

Cell-free DNA (cfDNA) has the potential to inform tumor subtype classification and help guide clinical precision oncology. Here we develop Griffin, a framework for profiling nucleosome protection and accessibility from cfDNA to study the phenotype of tumors using as low as 0.1x coverage whole genome sequencing data. Griffin employs a GC correction procedure tailored to variable cfDNA fragment sizes, which generates a better representation of chromatin accessibility and improves the accuracy of cancer detection and tumor subtype classification. We demonstrate estrogen receptor subtyping from cfDNA in metastatic breast cancer. We predict estrogen receptor subtype in 139 patients with at least 5% detectable circulating tumor DNA with an area under the receive operator characteristic curve (AUC) of 0.89 and validate performance in independent cohorts (AUC = 0.96). In summary, Griffin is a framework for accurate tumor subtyping and can be generalizable to other cancer types for precision oncology applications.

Importance DNA damage repair (DDR) gene mutations represent actionable alterations that can guide precision medicine strategies for advanced prostate cancer. However, acquisition of contemporary tissue samples for molecular testing can be a barrier to deploying precision medicine approaches. We hypothesized that most DDR alterations represent truncal events in prostate cancer and that primary tissue would faithfully reflect mutations found in cell-free circulating tumor DNA (ctDNA) and/or metastatic tissue. Objective To assess concordance in DDR gene alterations between primary prostate cancer and metastases or ctDNA specimens. Design, Setting, and Participants Patients were included if a DDR pathway mutation was detected in metastatic tissue or ctDNA and primary tissue sequencing was available for comparison. Sequencing data from 3 cohorts were analyzed: (1) FoundationOne, (2) University of Washington clinical cases (University of Washington–OncoPlex or Stand Up to Cancer–Prostate Cancer Foundation International Dream Team sequencing pipelines), and (3) University of Washington rapid autopsy series. Only pathogenic somatic mutations were included, and more than 30 days between primary tumor tissue and ctDNA and/or metastatic tissue acquisition was required. Clonal hematopoiesis of indeterminate potential (CHIP) and germline events were adjudicated by an expert molecular pathologist and excluded. Main Outcomes and Measures The DDR gene alterations detected in primary prostate tissue matched with metastatic tissue and/or ctDNA findings. Results A total of 72 men with known DDR alterations were included in the analysis, and primary samples with paired ctDNA and/or metastatic tissue were sequenced. After excluding patients with ctDNA where only CHIP and/or germline events (n = 21) were observed, 51 patients remained and were included in the final analysis. The median (range) time from acquisition of primary tissue to acquisition of ctDNA or tumor tissue was 55 (5-193) months. Concordance in DDR gene mutation status across samples was 84% (95% CI, 71%-92%). Rates of concordance between metastatic-primary and ctDNA-primary pairs were similar when patients with CHIP events were excluded. MulticlonalBRCA2reversion mutations associated with resistance to PARP inhibitors and platinum chemotherapy were detected in ctDNA from 2 patients. Conclusions and Relevance In this genetic association study of 3 patient cohorts, primary prostate tissue accurately reflected the mutational status of actionable DDR genes in metastatic tissue, consistent with DDR alterations being truncal in most patients. After excluding likely CHIP events, ctDNA profiling accurately captured these DDR mutations while also detecting reversion alterations that may suggest resistance mechanisms.

Prostate cancer (PC) is driven by androgen receptor (AR) activity, a master regulator of prostate development and homeostasis. Frontline therapies for metastatic PC deprive the AR of the activating ligands testosterone (T) and dihydrotestosterone (DHT) by limiting their biosynthesis or blocking AR binding. Notably, AR signaling is dichotomous, inducing growth at lower activity levels, while suppressing growth at higher levels. Recent clinical studies have exploited this effect by administration of supraphysiological concentrations of T, resulting in clinical responses and improvements in quality of life. However, the use of T as a therapeutic agent in oncology is limited by poor drug-like properties as well as rapid and variable metabolism. Here, we investigated the antitumor effects of selective AR modulators (SARMs), which are small-molecule nonsteroidal AR agonists developed to treat muscle wasting and cachexia. Several orally administered SARMs activated the AR program in PC models. AR cistromes regulated by steroidal androgens and SARMs were superimposable. Coregulatory proteins including HOXB13 and GRHL2 comprised AR complexes assembled by both androgens and SARMs. At bioavailable concentrations, SARMs repressed MYC oncoprotein expression and inhibited the growth of castration-sensitive and castration-resistant PC in vitro and in vivo. These results support further clinical investigation of SARMs for treating advanced PC.

INTRODUCTION AND OBJECTIVES:Although the genomic landscape of LTUC is well studied, less is known about UTUC, including in the metastatic sites. We evaluated and compared genomic features of metast...

Background: Cell-free DNA (cfDNA) is released from dying cells, including tumor cells, and can be isolated from peripheral blood for studying cancer. In the bloodstream, cfDNA is protected from degradation by nucleosomes and other DNA binding proteins, leading to a coverage pattern that reflects the genomic organization in the cells-of-origin. Recent work has shown that it is possible to use this pattern to predict gene and transcription factor activity in cancer cells. This is known as nucleosome profiling. Breast cancer is among the most common causes of cancer, accounting for 23% of cancer diagnoses and 14% of cancer-related deaths among women worldwide. Targeted therapy is guided by tumor subtype, including the expression of three key receptors: ER, PR and HER2. Typically, subtyping involves a tumor biopsy and immunohistochemistry. However, in late-stage cancer, surgical biopsies for disease monitoring are difficult to obtain. Accurate subtype determination is critical to address hormone subtype switches during metastasis or treatment resistance. cfDNA offers an alternative, non-invasive method for identifying tumor subtypes through nucleosome profiling and, to the best of our knowledge, has not been shown for breast cancer. Methods: We developed a method, called Griffin, to examine nucleosome protection and genome accessibility by quantifying cfDNA fragments around accessible sites. Unlike previous methods, Griffin uses fragment length-based GC correction to remove GC biases that obscure signals. We used ATAC-seq data from TCGA to identify differentially accessible sites between ER positive and negative breast cancers. We developed a machine learning classifier that predicts ER subtype based upon the signals at these differentially accessible sites. Results: We then tested Griffin by examining differentially accessible sites in ultra-low pass sequencing (ULP-WGS, 0.1X) of several hundred cfDNA samples from patients with ER positive or negative breast cancer. We found that overall, differential sites were more accessible in the cfDNA of their respective subtypes. Additionally, we found that site accessibility within patient cfDNA samples was correlated to the cfDNA tumor fraction. We built and tested a prediction model with cross-validation, which revealed an accuracy of >80% for correctly classifying tumor status as ER positive or negative from this ULP-WGS dataset. Conclusion: This study has several novel aspects compared to prior nucleosome profiling approaches. First, we use fragment-based GC correction which reduces sample variability and allows us to observe previously obscured signals. Second, we demonstrated that signals are correlated to tumor fraction. And finally, we applied this method to cost-effective and scalable ULP-WGS of breast cancer and demonstrated the ability to predict breast cancer ER subtype in these samples. Citation Format: Anna-Lisa Doebley, Hanna Liao, Caroline Kikawa, Eden Cruikshank, Minjeong Ko, Anna Hoge, Joseph Hiatt, Navonil De Sarkar, Viktor A. Adalsteinsson, Paz Polak, David MacPherson, Peter S. Nelson, Heather A. Parsons, Daniel Stover, Gavin Ha. Griffin: A method for nucleosome profiling and breast cancer subtype prediction from ultra-low pass whole genome sequencing of cell-free DNA [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB022.

Introduction: Exceedingly little is known about the genomic difference between metastatic urothelial carcinoma (LTUC) and upper tract urothelial carcinoma (UTUC). We evaluated and compared genomic features of primary and metastatic UTUC and LTUC tumors in a cohort of patients with end-stage disease. Methods: We performed whole-exome sequencing on 37 tumor samples from 7 patients with metastatic UC collected between 2015-2017 via rapid autopsy, with matched primary and metastatic tumor samples. Inter- and intrapatient analyses of mutational burden, mutational signatures, predicted deleterious mutations (somatic single nucleotide variations [sSNV] and insertions-deletions), and somatic copy alterations (sCNV) were conducted. Results: We investigated 3 patients with UTUC (3 primary samples, 13 metastases) and 4 patients with LTUC (4 primary samples, 17 metastases). Most patients were men, nonsmokers, and received cisplatin-based therapy. We found that sSNV burden was higher in metastatic LTUC compared to UTUC. Moreover, the APOBEC mutational signature that has been described in localized bladder cancer was pervasive in metastatic LTUC and less so in UTUC. Despite a lower overall sSNV burden, UTUC displayed greater inter- and intraindividual genomic distances at the copy number level between primary and metastatic tumors than LTUC. Our data also indicate that metastatic UTUC lesions can arise from small clonal populations present in the primary cancer. Importantly, putative druggable mutations were found across our patient population with the majority shared across all metastases within a patient. Conclusions: In these patients, metastatic UTUC demonstrated a lower overall mutational burden but greater structural variability compared to LTUC. Our findings suggest that metastatic UTUC displays a greater spectrum of copy number divergence from LTUC, which may in part explain differences in aggressive clinical behavior. Importantly, we identified druggable lesions shared across metastatic samples, which demonstrate a level of targetable homogeneity within individual patients. This abstract is also being presented as Poster B09. Citation Format: Brian R. Winters, Navonil De Sarkar, Sonali Arora, Hamid Bolouri, Sujata Jana, Funda Vakar-Lopez, Heather H. Cheng, Michael T. Schweizer, Evan Y. Yu, Petros Grivas, John K. Lee, Lori Kollath, Sarah K. Holt, Lisa McFerrin, Gavin Ha, Peter S. Nelson, Robert B. Montgomery, Jonathan L. Wright, Hung-Ming Lam, Andrew C. Hsieh. Genomic distinctions between metastatic lower and upper tract urothelial carcinoma revealed through rapid autopsy [abstract]. In: Proceedings of the AACR Special Conference on Bladder Cancer: Transforming the Field; 2019 May 18-21; Denver, CO. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(15_Suppl):Abstract nr PR06.

BACKGROUND Plasma-based cell-free DNA is an attractive biospecimen for assessing somatic mutations due to minimally-invasive real-time sampling. However, next generation sequencing (NGS) of cell-free DNA (cfDNA) may not be appropriate for all patients with advanced prostate cancer (PC). METHODS Blood was obtained from advanced PC patients for plasma-based sequencing. UW-OncoPlex, a ∼2 Mb multi-gene NGS panel performed in the CLIA/CAP environment, was optimized for detecting cfDNA mutations. Tumor tissue and germline samples were sequenced for comparative analyses. Multivariate logistic regression was performed to determine the clinical characteristic associated with the successful detection of somatic cfDNA alterations (ie detection of at least one clearly somatic PC mutation). RESULTS Plasma for cfDNA sequencing was obtained from 93 PC patients along with tumor tissue (N = 67) and germline (N = 93) controls. We included data from 76 patients (72 prostate adenocarcinoma; 4 variant histology PC) in the analysis. Somatic DNA aberrations were detected in 34 cfDNA samples from patients with prostate adenocarcinoma. High PSA level, high tumor volume, and castration-resistance were significantly associated with successful detection of somatic cfDNA alterations. Among samples with somatic mutations detected, the cfDNA assay detected 93/102 (91%) alterations found in tumor tissue, yielding a clustering-corrected sensitivity of 92% (95% confidence interval 88-97%). All germline pathogenic variants present in lymphocyte DNA were also detected in cfDNA (N = 12). Somatic mutations from cfDNA were detected in 30/33 (93%) instances when PSA was >10 ng/mL. CONCLUSIONS Disease burden, including a PSA >10 ng/mL, is strongly associated with detecting somatic mutations from cfDNA specimens.

Significance The genomic landscape of metastatic castration-resistant prostate cancer (mCRPC) has been well-defined, but the association of genomic findings with patient clinical outcomes and with other characteristics including histology and transcriptional pathway activity remains poorly understood. Here, we describe comprehensive integrative analysis of genomic and transcriptomic profiles, histology, and clinical outcomes for 429 patients with mCRPC. Of all the molecular factors we examined, alterations in RB1 had the strongest association with poor outcome. Our study identifies molecularly defined groups of patients who may benefit from a more aggressive treatment approach, with the genomic and outcome data made available to the research community for further interrogation., Heterogeneity in the genomic landscape of metastatic prostate cancer has become apparent through several comprehensive profiling efforts, but little is known about the impact of this heterogeneity on clinical outcome. Here, we report comprehensive genomic and transcriptomic analysis of 429 patients with metastatic castration-resistant prostate cancer (mCRPC) linked with longitudinal clinical outcomes, integrating findings from whole-exome, transcriptome, and histologic analysis. For 128 patients treated with a first-line next-generation androgen receptor signaling inhibitor (ARSI; abiraterone or enzalutamide), we examined the association of 18 recurrent DNA- and RNA-based genomic alterations, including androgen receptor (AR) variant expression, AR transcriptional output, and neuroendocrine expression signatures, with clinical outcomes. Of these, only RB1 alteration was significantly associated with poor survival, whereas alterations in RB1, AR, and TP53 were associated with shorter time on treatment with an ARSI. This large analysis integrating mCRPC genomics with histology and clinical outcomes identifies RB1 genomic alteration as a potent predictor of poor outcome, and is a community resource for further interrogation of clinical and molecular associations.

Background: Gallbladder carcinoma (GBC) is a subtype of biliary tract malignancy with poor prognosis and high fatality rate. The present study was designed to uncover somatic and rare germline mutations in GBC to reveal the disease biology and understand the clinical importance of mutation profile in terms of prognostics and actionability. Materials and Methods: We performed ultra-deep sequencing across 409 cancer-related genes in 11 GBC patients of North-Indian descent. NGS data analysis was performed using Ion Reporter and several other publicly available resources and databases. Results: We identified 184 nonsynonymous somatic and 60 rare germline mutations in bona-fide cancer drivers such as SMAD family member 4 (SMAD4), lysine methyltransferase 2C (KMT2C), and tumor protein p53 (TP53). All the early-onset cases or hypermutated cases harbored mutation(s) in critical DNA-repair genes. Additionally, we detected 9 novel genes with high-impact somatic mutations in GBC. Conclusion: Our results indicated the significance of inherited rare germline mutations in DNA-repair pathway genes in addition to acquired somatic mutations in GB carcinogenesis.

Oral cancer generally progresses from precancerous lesions such as leukoplakia (LK), lichen planus (LP) and oral submucous fibrosis (OSMF). Since few of these precancers progress to cancers; it is worth to identify biological molecules that may play important roles in progression. Here, expression deregulation of 7 miRNAs (mir204, mir31, mir31*, mir133a, mir7, mir206 and mir1293) and their possible target genes in 23 cancers, 18 LK, 12 LP, 23 OSMF tissues compared to 20 healthy tissues was determined by qPCR method. Expression of mir7, mir31, mir31* and mir1293 was upregulated and that of mir133a, mir204 and mir206 was downregulated in cancer. Expression of most of these miRNAs was also upregulated in LK and LP tissues but not in OSMF. Expression deregulation of some of the target genes was also determined in cancer, LK and LP tissues. Significant upregulation of mir31 and downregulation of its target gene, CXCL12, in cancer, LK and LP tissues suggest their importance in progression of precancer to cancer. Expression upregulation of mir31 was also validated using GEO data sets. Although sample size is low, novelty of this work lies in studying expression deregulation of miRNAs and target genes in oral cancer and three types of precancerous lesions.

Mismatches at the 3'end of /or within a primer are reported to affect the efficiency of PCR and cause allele drop. Here, we report preferential amplification of one haplotype and misgenotyping, when double heterozygotes at NAT1 (rs1057126 and rs15561) were genotyped by sequencing and PCR-RFLP methods using mismatch reverse primers located next to the target SNP. Detailed study revealed highest (100%) and lowest (0%) misgenotyping when the mismatch was at the 3rd and 15th nucleotide positions from 3' end of the primer, respectively. But, the same primers, without any mismatch genotyped heterozygotes correctly. Homozygotes can always be detected correctly irrespective of mismatch position in the primer. Similar results were observed for two SNPs (rs12947788 and rs 12951053) at TP53. Using mismatch NAT1 reverse primers, located three nucleotides away from the target SNP, both TaqMan and sequencing methods showed preferential synthesis of one haplotype strand and misgenotyping in heterozygotes, respectively. So, mismatch primer, located next to target SNP, should be avoided to genotype heterozygotes, since, PCR and sequencing based genotyping methods may lead the investigators to report faulty allelic and genotypic frequencies. This study mimics a situation when an unknown variation is present in the primer-binding sites of both chromosomes.

371 Background: Although the genomic landscape of LTUC is well studied, less is known about UTUC, including in the metastatic sites. We compared genomic features of metastatic UTUC and LTUC. Methods: We performed whole exome sequencing on 7 rapid autopsy patients with metastatic UC, with matched primary and metastatic tumor samples (N = 37). Single nucleotide variants (SNV) were identified using Mutect and Strelka. Focused analyses were performed on mutations with known significance in UC as well as mutations predicted to have functional impact using 11 mutation assessors. Genome scale copy number aberrations (CNA) were estimated using Sequenza (normalized for ploidy) to derive gene definition restricted copy number estimation outcomes. Multi-dimensional scaling (MDS) was used to visualize how copy number and mutation-derived genomic distances differed between LTUC and UTUC. Results: Three pts with UTUC (3 primary samples, 13 metastases) and four pts with LTUC (4 primary samples, 17 metastases) were examined. The majority of patients were male (5) and received cisplatin-based therapy (5). We found that SNV burden (mean mutation per megabase) was significantly higher in LTUC vs. UTUC overall (6.6 vs. 3.8, p = 0.001) and when stratified by primaries (6.1 vs. 2.9, p = 0.047); or metastases (6.7 vs. 4.1, p = 0.001). Mutational signature analysis revealed higher proportion of APOBEC signature in all LTUC vs. UTUC tumors. Both inter- and intra-individual genomic distances between primary and metastatic tissues were substantially larger in UTUC than LTUC, suggesting a wider spectrum of mutations at the level of individual nucleotides and chromosomal structure. Interestingly, Gene definition-restricted CNA analysis revealed MDM2 amplification exclusively in UTUC tumors which was associated with shallow p53 deletion. Conclusions: Metastatic UTUC appears to have a lower overall mutational burden but greater genomic variability compared to LTUC. Our relatively small dataset suggests that metastatic UTUC displays a greater spectrum of mutational divergence from LTUC which may partially explain differences in clinical behavior.

Introduction: Prostate cancer (PC) exhibits a spectrum of genomic aberrations, a subset of which associate with responses to specific therapeutics. In particular, mutations in genes involved in DNA repair processes expose therapeutic vulnerabilities that include PARP inhibitors and DNA-damaging chemotherapy for homology-directed repair(HR) defects and immune checkpoint blockade for mismatch repair (MMR) defects. Deploying appropriate therapy is dependent on accurate tumor classification. Experimental Procedures: We evaluated whole-exome sequences derived from localized (n = 465) and metastatic (n = 208) PCs to identify mutations and structural aberrations predicted to disrupt or alter the function of genes involved in DNA repair processes. We annotated all tumors for trinucleotide (COSMIC) mutation signatures by comparing somatic alterations in tumor versus paired normal exomes. We compared the type and frequency of presumed pathogenic mutations, tumor ploidy, copy number aberrations, and mutational signatures between primary and metastatic tumors and evaluated associations between specific DNA repair gene aberrations and mutation signatures. Results: Overall, 54% of localized PCs and 24.5% of metastatic PCs had a component of their somatic mutation burden associated with a mutation signature indicative of a DNA repair defect. Metastatic PCs had significantly higher numbers of somatic mutations (n=223) compared to primary PCs (n=93) (p0.1) scores were enriched for pathogenic mutations in BRCA1/2 and lowest LOH scores were observed in tumors with mutation/loss of CDK12, which were near-diploid. In addition to relatively frequent aberrations in BRCA2, BRCA1 and ATM, we found CDK12 mutations in ~1% of primary PCs and 5.3% of metastatic PCs. Tumors with BRCA1/2 loss or ATM and RecQ-Helicase pathway aberration exhibited larger amplifications and loss events, whereas PCs with CDK12 aberrations demonstrated a high number (n>73 to 261) of focal (500b-1mb range) amplifications with rare focal losses. Conclusions: 102 individual metastatic cases with some fraction of HR-defect signature and 41 (~40%) of them are without any bona fide HR pathway mutations. DNA repair gene (DRG) aberrations are common in metastatic PC and associate with distinct mutational signatures and structural aberrations that partition PCs into distinct subtypes. Epigenetic processes may contribute to HR signatures in tumors where DRG mutations were not identified. Preclinical and clinical studies are required to determine if mutation signatures are accurate predictors of responses to therapeutics. Thus, with these signatures, we have proposed a few molecular subcategories of prostate tumors and such information can be harnessed for future precision cancer therapies. Citation Format: Navonil De Sarkar, Emily Kohlbrenner, Ilsa Coleman, Peter S. Nelson. Defining prostate cancer subtypes by genomic features associated with defects in DNA repair processes [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 424.

Using integrative genomic analysis of 360 metastatic castration-resistant prostate cancer (mCRPC) samples, we identified a novel subtype of prostate cancer typified by biallelic loss of CDK12 that is mutually exclusive with tumors driven by DNA repair deficiency, ETS fusions, and SPOP mutations. CDK12 loss is enriched in mCRPC relative to clinically localized disease and characterized by focal tandem duplications (FTDs) that lead to increased gene fusions and marked differential gene expression. FTDs associated with CDK12 loss result in highly recurrent gains at loci of genes involved in the cell cycle and DNA replication. CDK12 mutant cases are baseline diploid and do not exhibit DNA mutational signatures linked to defects in homologous recombination. CDK12 mutant cases are associated with elevated neoantigen burden ensuing from fusion-induced chimeric open reading frames and increased tumor T cell infiltration/clonal expansion. CDK12 inactivation thereby defines a distinct class of mCRPC that may benefit from immune checkpoint immunotherapy.

Several studies reported that mtDNA mutations may play important roles in carcinogenesis although the mechanism is not clear yet. Most of the studies compared mtDNA sequences in a tumor with those in normal tissues from different individuals ignoring inter-individual variations. In this study, 271 SNPs, 7 novel SNPs (or SNVs), and 15 somatic mutations were detected in mtDNA of 8 oral cancer tissues with respect to reference (rCRS) and adjacent normal tissues, respectively, using Ion PGM next generation sequencing method. Most of the sequence variations (76 SNPs and 1 somatic) are present in D-loop region followed by CyB (36 SNPs), ATP6 (24 SNPs), ND5 (17 SNPs and 5 somatic), ND4 (18 coding and 2 somatic) and other non-coding and coding DNA sequences. A total of 53 and 8 non-synonymous SNPs and somatic mutations, respectively, were detected in tumor tissues and some of these variations may have deleterious effects on the protein function as predicted by bioinformatic analysis. Moreover, significantly low mtDNA contents and expression of several mitochondrial genes in tumor compared to adjacent normal tissues may have also affected mitochondrial functions. Taken together, this study suggests that mtDNA mutations as well as low expression of mtDNA coded genes may play important roles in tumor growth. Although the sample size is low, an important aspect of the study is the use of adjacent control tissues to find out somatic mutations and a change in the expression of mitochondrial genes, to rule out inter-individual and inter-tissue variations which are important issues in the study of mitochondrial genomics.

Inherited mutations in DNA-repair genes such as BRCA2 are associated with increased risks of lethal prostate cancer. Although the prevalence of germline mutations in DNA-repair genes among men with localized prostate cancer who are unselected for family predisposition is insufficient to warrant routine testing, the frequency of such mutations in patients with metastatic prostate cancer has not been established.We recruited 692 men with documented metastatic prostate cancer who were unselected for family history of cancer or age at diagnosis. We isolated germline DNA and used multiplex sequencing assays to assess mutations in 20 DNA-repair genes associated with autosomal dominant cancer-predisposition syndromes.A total of 84 germline DNA-repair gene mutations that were presumed to be deleterious were identified in 82 men (11.8%); mutations were found in 16 genes, including BRCA2 (37 men [5.3%]), ATM (11 [1.6%]), CHEK2 (10 [1.9% of 534 men with data]), BRCA1 (6 [0.9%]), RAD51D (3 [0.4%]), and PALB2 (3 [0.4%]). Mutation frequencies did not differ according to whether a family history of prostate cancer was present or according to age at diagnosis. Overall, the frequency of germline mutations in DNA-repair genes among men with metastatic prostate cancer significantly exceeded the prevalence of 4.6% among 499 men with localized prostate cancer (P0.001), including men with high-risk disease, and the prevalence of 2.7% in the Exome Aggregation Consortium, which includes 53,105 persons without a known cancer diagnosis (P0.001).In our multicenter study, the incidence of germline mutations in genes mediating DNA-repair processes among men with metastatic prostate cancer was 11.8%, which was significantly higher than the incidence among men with localized prostate cancer. The frequencies of germline mutations in DNA-repair genes among men with metastatic disease did not differ significantly according to age at diagnosis or family history of prostate cancer. (Funded by Stand Up To Cancer and others.).

Oral cancer is usually preceded by pre-cancerous lesion and related to tobacco abuse. Tobacco carcinogens damage DNA and cells harboring such damaged DNA normally undergo apoptotic death, but cancer cells are exceptionally resistant to apoptosis. Here we studied association between sequence and expression variations in apoptotic pathway genes and risk of oral cancer and precancer. Ninety nine tag SNPs in 23 genes, involved in mitochondrial and non-mitochondrial apoptotic pathways, were genotyped in 525 cancer and 253 leukoplakia patients and 538 healthy controls using Illumina Golden Gate assay. Six SNPs (rs1473418 at BCL2; rs1950252 at BCL2L2; rs8190315 at BID; rs511044 at CASP1; rs2227310 at CASP7 and rs13010627 at CASP10) significantly modified risk of oral cancer but SNPs only at BCL2, CASP1and CASP10 modulated risk of leukoplakia. Combination of SNPs showed a steep increase in risk of cancer with increase in "effective" number of risk alleles. In silico analysis of published data set and our unpublished RNAseq data suggest that change in expression of BID and CASP7 may have affected risk of cancer. In conclusion, three SNPs, rs1473418 in BCL2, rs1950252 in BCL2L2 and rs511044 in CASP1, are being implicated for the first time in oral cancer. Since SNPs at BCL2, CASP1 and CASP10 modulated risk of both leukoplakia and cancer, so, they should be studied in more details for possible biomarkers in transition of leukoplakia to cancer. This study also implies importance of mitochondrial apoptotic pathway gene (such as BCL2) in progression of leukoplakia to oral cancer.

Introduction: Oral cancer is one of the leading causes of cancer burden in India. Studies over the years have enforced the role of deregulated microRNA expression in oral cancer development and progression. Incidentally not much is known about the cause of microRNA deregulation or to what extent can it be attributed to methylation. Established genome wide microarray platforms like 450K Illumina Bead Chip have probes for very limited number of microRNAs along with restriction on distance of the probes from microRNA transcription start site. We have used genome wide methylation sequencing approach RRBS to get deeper insight on microRNA methylation. Aim: Genome wide methylation profile of 15 paired oral cancer and adjacent normal tissue was compared to generate a detailed map of microRNA methylation in oral cancer. The methylation results were further correlated to microRNA expression data generated on a subset of these samples. Methodology: RRBS based global methylation sequencing of 15 pairs of samples was conducted on HiSeq 2500 with an average 10X depth. Analyses were done using DMAP software. Previously generated TLDA based microRNA expression data was used for correlation analysis. Result: Similar to protein coding genes, microRNAs also showed methylation based clustering. Interestingly majority of methylation hits were more than 2kb upstream of miRNA transcription start site which is generally not considered in known microarrays. Distribution of hypo and hyper methylation sites is different across the various CpG features like cores and shelves as well as across chromosomes. MicroRNAs like mir-181c, miR-99b, miR-23a among others showed significant negative correlation between methylation frequency and expression level. IPA based pathway analysis emphasized repression of apoptosis and cell death pathways along with activation of cell proliferation and colony formation. Conclusion: Understanding the role of methylation in microRNA deregulation could help us detect and possibly prevent a chain of events, which lead to disrupted pathways eventually leading to cancer, at the onset. Note: This abstract was not presented at the meeting. Citation Format: Roshni Roy, Aniruddha Chatterjee, Navonil De Sarkar, Bidyut Roy. Genome wide study reveal microrna based methylation phenotype in oral 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 1484. doi:10.1158/1538-7445.AM2017-1484

Deregulation of miRNA expression may contribute to tumorigenesis and other patho-physiology associated with cancer. Using TLDA, expression of 762 miRNAs was checked in 18 pairs of gingivo buccal cancer-adjacent control tissues. Expression of significantly deregulated miRNAs was further validated in cancer and examined in two types of precancer (leukoplakia and lichen planus) tissues by primer-specific TaqMan assays. Biological implications of these miRNAs were assessed bioinformatically. Expression of hsa-miR-1293, hsa-miR-31, hsa-miR-31* and hsa-miR-7 were significantly up-regulated and those of hsa-miR-206, hsa-miR-204 and hsa-miR-133a were significantly down-regulated in all cancer samples. Expression of only hsa-miR-31 was significantly up-regulated in leukoplakia but none in lichen planus samples. Analysis of expression heterogeneity divided 18 cancer samples into clusters of 13 and 5 samples and revealed that expression of 30 miRNAs (including the above-mentioned 7 miRNAs), was significantly deregulated in the cluster of 13 samples. From database mining and pathway analysis it was observed that these miRNAs can significantly target many of the genes present in different cancer related pathways such as "proteoglycans in cancer", PI3K-AKT etc. which play important roles in expression of different molecular features of cancer. Expression of hsa-miR-31 was significantly up-regulated in both cancer and leukoplakia tissues and, thus, may be one of the molecular markers of leukoplakia which may progress to gingivo-buccal cancer.

Genetic variations at microRNA and microRNA processing genes are known to confer risk of cancer in different populations. Here, we studied variations at eight microRNA (miRNA) and four miRNA processing genes in 452 controls and 451 oral cancer patients by TaqMan genotyping assays. Variant allele-containing genotypes at mir-196a2 and variant allele homozygous genotype at Ran increased the risk of cancer significantly [adjusted odds ratio (OR) (95% confidence interval (CI)) = 1.3 (1-1.7) and 2.3 (1.1-4.6), respectively]. Conversely, variant allele-containing genotypes at mir-34b and variant allele homozygous genotype at Gemin3 reduced the risk of cancer significantly [adjusted OR (95% CI) = 0.7 (0.5-0.9) and 0.6 (0.4-1), respectively]. Cumulative risk was also increased by three times with increase in the number of risk alleles at these four loci. In tobacco stratified analysis, variant allele homozygous genotypes at mir-29a and Ran increased [adjusted OR (95% CI) = 1.5 (1-2.3) and 3 (1.1-8.4) respectively], while variant allele-containing genotypes at mir-34b decreased [adjusted OR (95% CI) = 0.6 (0.4-0.9)] the risk of cancer significantly. Thus, genetic variation at miRNA and processing genes altered the risk of oral cancer in this population thereby corroborating studies in other populations. However, it is necessary to validate this result in different Indian sub populations with larger sample sizes and examine the effect of these variations in tumour tissues to explain the mechanism of risk alteration.

5009Background: Inherited mutations in genes that regulate DNA repair processes such as BRCA1 and BRCA2 associate with increased risks of developing lethal prostate cancer (PC), and with response t...

MicroRNAs (miRNAs) which are on average only 21-25 nucleotides long are key post-transcriptional regulators of gene expression in metazoans and plants. A proper quantitative understanding of miRNAs is required to comprehend their structures, functions, evolutions etc. In this paper, the nucleotide strings of miRNAs of three organisms namely Homo sapiens (hsa), Macaca mulatta (mml) and Pan troglodytes (ptr) have been quantified and classified based on some characterizing features. A network has been built up among the miRNAs for these three organisms through a class of discrete transformations namely Integral Value Transformations (IVTs), proposed by Sk. S. Hassan et al [1, 2]. Through this study we have been able to nullify or justify one given nucleotide string as a miRNA. This study will help us to recognize a given nucleotide string as a probable miRNA, without the requirement of any conventional biological experiment. This method can be amalgamated with the existing analysis pipelines, for small RNA sequencing data (designed for finding novel miRNA). This method would provide more confidence and would make the current analysis pipeline more efficient in predicting the probable candidates of miRNA for biological validation and filter out the improbable candidates.

Background MicroRNAs have been implicated in cancer but studies on their role in precancer, such as leukoplakia, are limited. Sequence variations at eight miRNA and four miRNA processing genes were studied in 452 healthy controls and 299 leukoplakia patients to estimate risk of disease. Results Genotyping by TaqMan assay followed by statistical analyses showed that variant genotypes at Gemin3 and mir-34b reduced risk of disease [OR = 0.5(0.3–0.9) and OR = 0.7(0.5–0.9) respectively] in overall patients as well as in smokers [OR = 0.58(0.3–1) and OR = 0.68(0.5–0.9) respectively]. Among chewers, only mir29a significantly increased risk of disease [OR = 1.8(1–3)]. Gene-environment interactions using MDR-pt program revealed that mir29a, mir34b, mir423 and Xpo5 modulated risk of disease (p

Introduction: Oral leukoplakia is a premalignant lesion that long has been considered to confer increased risk for the development of Oral Cancer which is highly prevalent in India. Oral lichen planus (OLP) on the other hand is a chronic inflammatory disease whose malignant potential is debatable. Since not all oral precancerous lesions progress to oral cancer, molecular markers like miRNA expression could be used to diagnose and predict progression of disease that cannot be detected histopathologically. Objective: Study of intra- and inter-variation in the expression of a few microRNAs (26a1, 423, 34b, 29a, 193a, 137 and 205) and microRNA processing (XPO5, GEMIN3 and RAN) genes in the three disease types, namely, oral cancer, leukoplakia and lichen planus. Methodology: Total RNA was extracted from paired disease and normal tissues from 15 oral cancer, 19 leukoplakia and 19 OLP patients. Expression of microRNAs and microRNA processing genes were studied using TaqMan assay. Subsequent statistical analyses were performed for interpretation of expression data. Results: Expression of mir 26a-1 and mir 137 genes was dysregulated >2 folds in 70% of the cancer samples while expression of remaining microRNAs and mir-processing genes were deregulated in a few samples. In leukoplakia, expression of mir-26a-1 was dysregulated >2 folds in 50% of the samples while dysregulation in expression of the remaining miRNAs was observed in 1/3rd of the patients. In OLP, there was an interesting trend of unidirectional up or down regulation in the expression of mir26a-1, mir29a, mir-34b and mir-137 genes. We also observed that the average expression of miRNA genes differed among the three patient groups. Conclusion: Deregulation in microRNA expression could not be correlated with individual's tobacco habits. Variation in expression of microRNA as well as miRNA processing genes was observed within individuals of the same patient group (i.e. cancer or leukoplakia or lichen planus) and between any two patients groups. There was correlation in expression of microRNAs which probably explains the unidirectional increase or decrease of miR expression in OLP. Finally, the mean expression level in the three types of disease tissues differed significantly. But expression of more miRNA genes in more samples would be necessary for molecular markers. Citation Format: Bidyut Roy, Roshni Roy, Navonil De Sarkar, Ranjan Rashni Paul. Expression of selected miRNA and miRNA processing genes in oral precancer and cancer tissues. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5274. doi:10.1158/1538-7445.AM2013-5274