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Prostate cancer (PCa) accounted for more than 34,000 deaths in US males [...]

Background: The combination of abiraterone acetate and prednisone (AA/P) is used to treat metastatic prostate cancer, but molecular predictors of treatment response are not well elucidated. We evaluated plasma circulating tumor DNA– (ctDNA-) based copy number alterations (CNAs) to determine treatment-related predictive and prognostic biomarkers for metastatic castration-resistant prostate cancer (mCRPC). Methods: Serial plasma specimens were prospectively collected from 88 chemotherapy-naive mCRPC patients before and after 12 weeks of AA/P treatment. Sequencing-based CNA analyses were performed on 174 specimens. We evaluated CNA-associated 12-week responses for primary resistance, time to treatment change (TTTC) for secondary resistance, and overall survival for prognosis (P < .05). Associations with primary resistance were analyzed using the Fisher exact test. Kaplan–Meier survival curves and Cox regression analyses were used to determine the associations of CNAs with acquired resistance and overall survival. Results: ctDNA reduced by 3.89% in responders and increased by 0.94% in nonresponders (P = .0043). Thirty-one prostate cancer–related genes from whole genome CNAs were tested. AR and AR enhancer amplification were associated with primary resistance (P = .0039) and shorter TTTC (P = .0003). ZFHX3 deletion and PIK3CA amplification were associated with primary resistance (P = .026 and P = .017, respectively), shorter TTTC (P = .0008 and P= .0016, respectively), and poor survival (P = .0025 and P = .0022, respectively). CNA-based risk scores combining selected significant associations (AR, NKX3.1, and PIK3CA) at the univariate level with TTTC were predictive of secondary resistance (P = .0002). and established prognoses for survival based on CNAs in ZFHX3, RB1, PIK3CA, and OPHN1 (P = .002). Multigene risk scores were more predictive than individual genes or clinical risk factors (P < .05). Conclusion: Plasma ctDNA CNAs and risk scores can predict mCRPC-state treatment and survival outcomes.

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Amplification of androgen receptor (AR) is a common genomic event in metastatic castration-resistant prostate cancer (mCRPC). To evaluate the prognostic value of the amplifications of specific loci in the AR gene in cell-free DNA, we developed a multiplex digital PCR (dPCR) assay that targeted AR enhancer (AR-En), AR exon 1 (AR-E1), AR exon 8 (AR-E8) and OPHN1 (downstream of AR). We selected three relatively stable genes, C2orf16, FAM111B, and GRIA3, as reference controls for copy number normalization. One hundred and eight mCRPC patients were recruited to test the association of specific AR loci amplification with clinical outcome. Using a normalized ratio &ge, 1.92 as cutoff, amplification of AR-En, AR-E1, AR-E8 and OPHN1 was observed in 28, 25, 24 and 19 of 108 mCRPC patients, respectively. Among the 41 patients with AR region amplification, 9 (21.9%) showed amplification at all four selected regions and 15 (36.6%) showed amplification at AR-En, AR-E1, and AR-E8. Six (14.6%) patients showed independent AR-En amplification, while the remaining 3 (7.3%) demonstrated AR-E8 amplification only. Kaplan&ndash, Meier analysis showed overall survival&rsquo, s association with the amplification of AR-En (p = 0.02, HR = 1.68 (1.07&ndash, 2.65)), AR-E8 (p = 0.02, HR = 1.78 (1.08&ndash, 2.92)) and AR-En-E8 (the combination of AR-En and AR-E8 (p = 0.009, HR = 1.77 (1.15&ndash, 2.73)). Multivariate models that included AR-En-E8 amplification and clinical factors significantly improved prognostic performance (p = 0.0001). With further validation, the multiplex dPCR assay may assist in prognostication of mCRPC patients.

Objectives To identify genomic variations in cell-free DNA (cfDNA) and evaluate their clinical utility in small cell lung cancer (SCLC). Materials and methods We performed whole genome sequencing using plasma cfDNAs derived from 24 SCLC patients for copy number variation (CNV) analysis, and targeted sequencing using 17 pairs of plasma cfDNA and their matched gDNA for mutation analysis. We defined somatic mutations by comparing cfDNA to its matched gDNA with 5% variant alleles as the cutoff for mutation calls. We applied Kaplan-Meier to correlate the genomic alterations with overall survival (OS) and progression-free survival (PFS). Results We observed widespread somatic copy-number alterations and mutations, including amplification of MYC at 8q24, FGF10 at 5p13, SOX2 at 3q26 and FGFR1 at 8p12, as well as deletion of TP53 at 17p13, RASSF1 at 3p21.3, RB1 at 13q14.2, FHIT at 3p14, and PTEN at 10q23. The most frequent mutations were genes involved in chromatin regulation (KMT2D, ARID1A, SETBP1 and PBRM1), PI3K/MTOR pathway(MTOR,PIK13G), Notch1 signalling pathway (NOTCH1), and DNA repair related gene ATRX. Kaplan-Meier analysis revealed poor OS and PFS in patients with somatic mutations in gene SETBP1 (P = 0.0061/0.0264, HR = 4.785/3.841, 95% CI = 2.014–28.25/1.286–16.58) and PBRM1 (P = 0.0276/0.0286, HR = 3.532/3.506, 95% CI = 1.275 to 25.34/1.26–24.87). Poor OS was also associated with somatic mutations in ATRX (P = 0.0099, HR = 4.024, 95% CI = 1.926–42.95), EP300 (P = 0.025/0.0622, HR = 3.382/2.891, 95% CI = 1.448–27.76/1.013-17.29), while poor PFS was associated with ATM mutation (P = 0.0038, HR = 4.604, 95% CI = 2.211–40.93). The mutation index produced by summing up the number of mutations in the five genes was significantly associated with the poor OS/PFS (P = 0.0185/0.0294) after adjusting the effect of the stage. Conclusions Our result supports blood plasma as a promising sample source for the genomic analysis in SCLC patients whose tumor tissues are scarcely available and demonstrates potential clinical utilities of cfDNA-based liquid biopsy for clinical management of this deadly disease.

The prognostic significance of plasma cell-free DNA (cfDNA) androgen receptor amplification (ARamp) in metastatic castration-resistant prostate cancer (mCRPC) compared with circulating tumor cell (CTC) counts is not known. As part of correlative aims of a prospective study in mCRPC, concurrent and serial collections of plasma and CTCs were performed. Specimen collections were performed at baseline after progression on androgen deprivation therapy and then 12 weeks later. QuantStudio3D digital PCR system was used to determine plasma cfDNA AR copy number variations and Cell search assay for enumerating CTC counts. Association of baseline cfDNA ARamp status/CTC counts with overall survival (OS) (primary goal) was evaluated using Kaplan–Meier method and log-rank test (p ≤ 0.05 for significance) and receiver operator curves (ROCs) for ARamp status and CTC counts ≥5. A multivariate analysis was performed using Cox regression models that included ARamp, CTC counts, and other clinical factors. ARamp was detected in 19/70 patients at baseline. At the time of analysis, 28/70 patients had died (median follow-up 806 days; interquartile range: 535–966). ARamp was associated with poor OS (2-year OS of 35% in ARamp vs. 71% in non-ARamp; log-rank p value ≤0.0001). Baseline CTC counts ≥5 (vs.

Functional characterization of disease-causing variants at risk loci has been a significant challenge. Here we report a high-throughput single-nucleotide polymorphisms sequencing (SNPs-seq) technology to simultaneously screen hundreds to thousands of SNPs for their allele-dependent protein-binding differences. This technology takes advantage of higher retention rate of protein-bound DNA oligos in protein purification column to quantitatively sequence these SNP-containing oligos. We apply this technology to test prostate cancer-risk loci and observe differential allelic protein binding in a significant number of selected SNPs. We also test a unique application of self-transcribing active regulatory region sequencing (STARR-seq) in characterizing allele-dependent transcriptional regulation and provide detailed functional analysis at two risk loci (RGS17 and ASCL2). Together, we introduce a powerful high-throughput pipeline for large-scale screening of functional SNPs at disease risk loci., Functional characterization of disease-causing variants at risk loci in cancer is challenging. Here, in prostate cancer the authors report a pipeline for high-throughput single-nucleotide polymorphisms sequencing (SNPs-seq) for large scale screening of functional SNPs at disease risk loci.

Extrachromosomal circular DNAs (eccDNAs) have been reported in most eukaryotes. However, little is known about the cell-free eccDNA profiles in circulating system such as blood. To characterize plasma cell-free eccDNAs, we performed sequencing analysis in 26 libraries from three blood donors and negative controls. We identified thousands of unique plasma eccDNAs in the three subjects. We observed proportional eccDNA increase with initial DNA input. The detected eccDNAs were also associated with circular DNA enrichment efficiency. Increasing the sequencing depth in an additional sample identified many more eccDNAs with highly heterogenous molecular structure. Size distribution of eccDNAs varied significantly from 31 bp to 19,989 bp. We found significantly higher GC content in smaller eccDNAs (500 bp) (p

To evaluate the clinical utility of cell-free DNA (cfDNA), we performed whole-genome sequencing to systematically examine plasma cfDNA copy number variations (CNVs) in a cohort of patients with colorectal cancer (CRC, n = 80), polyps (n = 20), and healthy controls (n = 35). We initially compared cfDNA yield in 20 paired serum-plasma samples and observed significantly higher cfDNA concentration in serum (median = 81.20 ng, range 7.18-500 ng·mL-1 ) than in plasma (median = 5.09 ng, range 3.76-62.8 ng·mL-1 ) (P < 0.0001). However, tumor-derived cfDNA content was significantly lower in serum than in matched plasma samples tested. With ~10 million reads per sample, the sequencing-based copy number analysis showed common CNVs in multiple chromosomal regions, including amplifications on 1q, 8q, and 5q and deletions on 1p, 4q, 8p, 17p, 18q, and 22q. Copy number changes were also evident in genes critical to the cell cycle, DNA repair, and WNT signaling pathways. To evaluate whether cumulative copy number changes were associated with tumor stages, we calculated plasma genomic abnormality in colon cancer (PGA-C) score by summing the most significant CNVs. The PGA-C score showed predictive performance with an area under the curve from 0.54 to 0.84 for CRC stages I-IV. Locus-specific copy number analysis identified nine genomic regions where CNVs were significantly associated with survival in stage III-IV CRC patients. A multivariate model using six of nine genomic regions demonstrated a significant association of high-risk score with shorter survival (HR = 5.33, 95% CI = 6.76-94.44, P < 0.0001). Our study demonstrates the importance of using plasma (rather than serum) to test tumor-related genomic variations. Plasma cfDNA-based tests can capture tumor-specific genetic changes and may provide a measurable classifier for assessing clinical outcomes in advanced CRC patients.

Molecular analysis of cell-free DNA (cfDNA) that circulates in plasma and other body fluids represents a “liquid biopsy” approach for non-invasive cancer screening or monitoring. The rapid development of sequencing technologies has made cfDNA a promising source to study cancer development and progression. Specific genetic and epigenetic alterations have been found in plasma, serum, and urine cfDNA and could potentially be used as diagnostic or prognostic biomarkers in various cancer types. In this review, we will discuss the molecular characteristics of cancer cfDNA and major bioinformatics approaches involved in the analysis of cfDNA sequencing data for detecting genetic mutation, copy number alteration, methylation change, and nucleosome positioning variation. We highlight specific challenges in sensitivity to detect genetic aberrations and robustness of statistical analysis. Finally, we provide perspectives regarding the standard and continuing development of bioinformatics analysis to move this promising screening tool into clinical practice.

Background: Chemopreventive agent (CPA) treatment is one of the main preventive options for lung cancer. However, few studies have been done on pharmacodynamic biomarkers of known CPAs for lung cancer. Materials and methods: In this study, we treated mouse models of lung squamous cell carcinoma with three different CPAs (MEK inhibitor: AZD6244, PI-3K inhibitor: XL-147 and glucocorticoid: Budesonide) and examined circulating exosomal miRNAs in the plasma of each mouse before and after treatment. Results: Compared to baselines, we found differentially expressed exosomal miRNAs after AZD6244 treatment (n = 8, FDR &lt, 0.05, n = 55, raw p-values &lt, 0.05), after XL-147 treatment (n = 4, FDR &lt, n = 26, raw p-values &lt, 0.05) and after Budesonide treatment (n = 1, FDR &lt, n = 36, raw p-values &lt, 0.05). In co-expression analysis, we found that modules of exosomal miRNAs reacted to CPA treatments differently. By variable selection, we identified 11, 9 and nine exosomal miRNAs as predictors for AZD6244, XL-147 and Budesonide treatment, respectively. Integrating all the results, we highlighted 4 miRNAs (mmu-miR-215-5p, mmu-miR-204-5p, mmu-miR-708-3p and mmu-miR-1298-5p) as the key for AZD6244 treatment, mmu-miR-23a-3p as key for XL-147 treatment, and mmu-miR-125a-5p and mmu-miR-16-5p as key for Budesonide treatment. Conclusions: This is the first study to use circulating exosomal miRNAs as pharmacodynamic biomarkers for CPA treatment in lung cancer.

Genetic profiling of urine cell free DNA (cfDNA) has not been evaluated in advanced prostate cancer. We performed whole genome sequencing of urine cfDNAs to identify tumor-associated copy number variations in urine before and after initiating androgen deprivation therapy in HSPC stage and docetaxel chemotherapy in CRPC stage. A log2 ratio-based copy number analysis detected common genomic abnormalities in prostate cancer including AR amplification in 5/10 CRPC patients. Other abnormalities identified included TMPRSS2-ERG fusion, PTEN gene deletion, NOTCH1 locus amplification along with genomic amplifications at 8q24.3, 9q34.3, 11p15.5 and 14q11.2, and deletions at 4q35.2, 5q31.3, 7q36.3, 12q24.33, and 16p11.2. By comparing copy number between pre- and post-treatment, we found significant copy number changes in 34 genomic loci. To estimate the somatic tumor DNA fraction in urine cfDNAs, we developed a Urine Genomic Abnormality (UGA) score algorithm that summed the top ten most significant segments with copy number changes. The UGA scores correlated with tumor burden and the change in UGA score after stage-specific therapies reflected disease progression status and overall survival. The study demonstrates the potential clinical utility of urine cfDNAs in predicting treatment response and monitoring disease progression.

Background: Androgen receptor (AR) amplification is a common genomic event in advanced prostate cancer, in particular, the metastatic castration-resistant prostate cancer (mCRPC) and is associated with poor treatment response and short overall survival (OS). Recent studies also show more frequent amplification at AR enhancer region in the mCRPC patients. The aim of this study is to evaluate clinical utility of multiplex digital PCR assay to detect amplification at AR locus in cell-free DNA (cfDNA) for prognostication of mCRPC. Methods: Plasma samples were collected from 108 patients with mCRPC before stage-specific treatment. The target regions were selected based on our previous sequencing data showing frequent amplification in mCRPC patients at AR loci, including AR enhancer, AR (AR exon 1 and exon 8) and OPHN1 (downstream of AR). Multiplex digital PCR assays to detect four targets were developed by adjusting the concentrations of primers and probes. The Kaplan-Meier (KM) estimate was used for OS analysis. Results: We first examined existing databases to determine relative stable genomic regions in prostate cancer. This analysis showed most stable regions in three gene loci including C2orf16 at 2p23.2, FAM111B at 11q12.1, and GRIA3 at Xq25, which were then used as reference controls for copy number normalization. We then designed two multiplex digital PCR assays to quantify copy number of selected targets. To separate two different targets with same fluorescence, we adjusted ratio of FAM-labeled probes with 1: 0.4 and HEX-labeled probes with 1:0.4. The ratio of FAM to HEX probes was 1:1.5 in final assays. The combination of four different targets in one reaction gave 9 possible clusters in the 2D amplitude space. We defined the amplification as the normalized ratio ≥ 1.8 between target and reference loci. By applying this cutoff in 108 mCRPC samples, we detected the amplification of AR-enhancer, AR and OPHN1 in 38, 34, and 25 samples, respectively. KM analysis showed the significant association of amplification in AR-enhancer (p=0.0053, HR=2.16[1.41-6.53]), AR (p=0.04, HR=1.74[1.04-4.03]) and OPHN1 (p=0.031, HR=1.91[1.07-5.53]) with OS. This result was consistent with whole genome sequencing data in the same group of patients. Conclusions: We established a highly sensitive multiplex digital PCR assay to detect copy number changes of AR, AR enhancer and OPHN1 in plasma cfDNA and demonstrated its potential clinical use as non-invasive testing tool for mCRPC prognosis. The easy-to-use and low-cost multiplex digital PCR assay covers multiple gene loci, which can aid in improving the sensitivity of detecting amplification at AR locus in the highly heterogeneous mCRPC patients. Citation Format: Meijun Du, Chiang-Ching Huang, Winston Tan, Manish Kohli, Liang Wang. Clinical utility of multiplex digital PCR to detect androgen receptor amplification in cell-free DNA for prognosis of metastatic castration-resistant prostate cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4319.

Liquid biopsies, examinations of tumor components in body fluids, have shown promise for predicting clinical outcomes. To evaluate tumor-associated genomic and genetic variations in plasma cell-free DNA (cfDNA) and their associations with treatment response and overall survival, we applied whole genome and targeted sequencing to examine the plasma cfDNAs derived from 20 patients with advanced prostate cancer. Sequencing-based genomic abnormality analysis revealed locus-specific gains or losses that were common in prostate cancer, such as 8q gains, AR amplifications, PTEN losses and TMPRSS2-ERG fusions. To estimate tumor burden in cfDNA, we developed a Plasma Genomic Abnormality (PGA) score by summing the most significant copy number variations. Cox regression analysis showed that PGA scores were significantly associated with overall survival (p < 0.04). After androgen deprivation therapy or chemotherapy, targeted sequencing showed significant mutational profile changes in genes involved in androgen biosynthesis, AR activation, DNA repair, and chemotherapy resistance. These changes may reflect the dynamic evolution of heterozygous tumor populations in response to these treatments. These results strongly support the feasibility of using non-invasive liquid biopsies as potential tools to study biological mechanisms underlying therapy-specific resistance and to predict disease progression in advanced prostate cancer.

// Meijun Du 1, * , Karthik V. Giridhar 2, * , Yijun Tian 1, 3 , Michael R. Tschannen 4 , Jing Zhu 1 , Chiang-Ching Huang 5 , Deepak Kilari 6 , Manish Kohli 2 and Liang Wang 1 1 Department of Pathology and MCW Cancer Center, Medical College of Wisconsin, Milwaukee, WI, USA 2 Department of Oncology, Mayo Clinic, Rochester, MN, USA 3 Department of Oncology, Tongji Hospital of Tongji Medical College, Huazhong University of Science and Technology, Wuhan, P.R. China 4 Human and Molecular Genetics Center, Medical College of Wisconsin, Milwaukee, WI, USA 5 Department of Biostatistics, University of Wisconsin, Milwaukee, WI, USA 6 Department of Oncology, Medical College of Wisconsin and Milwaukee VA Medical Center, Milwaukee, WI, USA * These authors have contributed equally to this work Correspondence to: Liang Wang, email: liwang@mcw.edu Manish Kohli, email: kohli.manish@mayo.edu Keywords: metastatic renal cell cancer, exosomal miRNA, overall survival, biomarker Received: March 28, 2017 Accepted: June 19, 2017 Published: July 22, 2017 ABSTRACT Plasma exosomal miRNAs were evaluated for prognosis in an initial set of 44 metastatic renal cell cancer (mRCC) patients by RNA sequencing. Among ~3.49 million mappable reads per patient, miRNAs accounted for 93.1% of the mapped RNAs. 227 miRNAs with high abundance were selected for survival analysis. Cox regression analysis identified association of 6 miRNAs with overall survival (OS) (P

Chromosome 8q24 locus contains regulatory variants that modulate genetic risk to various cancers including prostate cancer (PC). However, the biological mechanism underlying this regulation is not well understood. Here, we developed a chromosome conformation capture (3C)-based multi-target sequencing technology and systematically examined three PC risk regions at the 8q24 locus and their potential regulatory targets across human genome in six cell lines. We observed frequent physical contacts of this risk locus with multiple genomic regions, in particular, inter-chromosomal interaction with CD96 at 3q13 and intra-chromosomal interaction with MYC at 8q24. We identified at least five interaction hot spots within the predicted functional regulatory elements at the 8q24 risk locus. We also found intra-chromosomal interaction genes PVT1, FAM84B and GSDMC and inter-chromosomal interaction gene CXorf36 in most of the six cell lines. Other gene regions appeared to be cell line-specific, such as RRP12 in LNCaP, USP14 in DU-145 and SMIN3 in lymphoblastoid cell line. We further found that the 8q24 functional domains more likely interacted with genomic regions containing genes enriched in critical pathways such as Wnt signaling and promoter motifs such as E2F1 and TCF3. This result suggests that the risk locus may function as a regulatory hub by physical interactions with multiple genes important for prostate carcinogenesis. Further understanding genetic effect and biological mechanism of these chromatin interactions will shed light on the newly discovered regulatory role of the risk locus in PC etiology and progression.

Extracellular vesicles are selectively enriched in RNA that has potential as disease biomarkers. To systemically characterize circulating extracellular RNA (exRNA) profiles, we performed RNA sequencing analysis on plasma extracellular vesicles derived from 50 healthy individuals and 142 cancer patients. Of ~12.6 million raw reads for each individual, the number of mappable reads aligned to RNA references was ~5.4 million including miRNAs (~40.4%), piwiRNAs (~40.0%), pseudo-genes (~3.7%), lncRNAs (~2.4%), tRNAs (~2.1%) and mRNAs (~2.1%). By expression stability testing, we identified a set of miRNAs showing relatively consistent expression, which may serve as reference control for exRNA quantification. By performing multivariate analysis of covariance, we identified significant associations of these exRNAs with age, sex and different types of cancers. In particular, down-regulation of miR-125a-5p and miR-1343-3p showed an association with all cancer types tested (false discovery rate

Representative immunohistochemistry analysis of xenograft tumor tissues. Cells with active caspase-3 were shown in brown color. A, B and C are representative images for a lower fraction of apoptotic cells, a higher fraction of apoptotic cells, and negative controls, respectively. (PPTX 1067 kb)

Higher miR-375 suppresses cell proliferation in PC cells. Overexpression of miR-375 by transfection with 200 nM miR-375 mimic inhibited cell growth when compared to miRNA negative control in PC-3 and DU145 cells at 72Â h after transfection. (Pâ

Ancient DNA methodologies can be applied in the investigation of the genetics of extinct populations. A search for beta thalassemia mutations was performed on 49 Minoan individuals from the Bronze Age who were living in the island of Crete approximately 4000 Years Before Present (YBP). Standard precautionary measures were employed in the laboratory to ensure authenticity of the DNA extracted from the ancient bones, resulting in the successful analysis of DNA of 24 Minoans. DNA sequencing focused on the Intervening Sequence 1 (IVS-1) of the beta globin gene and its splicing junctions. 63% of the thalassemia mutations observed among modern Cretans reside in beta IVS-1. None of the Minoan individuals carried one of the IVS-1 mutations known to cause beta thalassemia; however, only one was expected to be observed if the average frequency of beta thalassemia heterozygotes in the Minoan population was the same with that of modern day Cretans (7.6%). One individual contained a C to G substitution in position 91 of the IVS-1, located 40 bp 5′ to the intron 1/exon 2 junction. Functional studies indicated that the mutation did not affect mRNA splicing or stability, and most likely represented an innocent single nucleotide polymorphism.

Background: Abiraterone acetate/prednisone (AA/P) is used for treatment of metastatic castration-resistant prostate cancer (mCRPC), but response is variable and there are no known molecular predictive factors. Methods: We prospectively collected germline and plasma specimens from 72 mCRPC patients progressing on androgen-deprivation therapy (ADT) before initiating AA/P treatment (baseline) and serially after 12 weeks of treatment. After extracting cell-free DNA (cfDNA), we performed copy number analysis using low pass whole-genome sequencing approach. The primary endpoints were to determine association with response and survival. Cox regression and Kaplan-Meier survival analyses were used to associate the CNV with overall survival (OS) and progression-free survival (PFS) with statistical significance at p≤0.05. For determining primary resistance to AA/P, responders and nonresponders were defined as patients showing no progression or progression after 12 weeks treatment. T-test was used to evaluate the CNV with AA/P response at 12 weeks. Results: Whole-genome sequencing generated approximately 8.0 million mappable sequence reads per subject (total plasma samples sequenced, n=144). By comparing cfDNA to germline control DNA, our copy number analysis showed significant genomic abnormalities in 37 of 144 plasma samples. We observed the most frequent amplification of 1q, 8q, Xq and deletion of 2p, 6q,10q, 13q. Among these CNVs, amplification of MYC at 8q24.21, AR at Xq12, deletion of FOXP1 at 3p14, PTEN at 10q23.31, RB1 at 13q14.2, TP53 at 17P13.1, and gene fusions (TMPRSS2:ERG) were consistent with previous reports from tumor tissue-based studies. Survival analysis showed significant association of a fragment deletion at 13q14-32 with both OS and PFS (FDR= 0.0207/0.0141). The most significant fragment showing association with OS was the deletion of fragment at 2p22.1-p14 (FDR=0.00219). Genomic gains of MYC at 8q24.21 and NCOA2 at 8q13.3 were associated with PFS with FDR= 0.0085 and 0.0072, respectively. By comparing baseline CNVs between responder and nonresponders, we identified significant association of the deletion of PTEN at 10q23.31 (p= 0.0003), RB1 at 13q14.2 (p= 0.0008) and amplification of NCOA2 at 8q13.3 (p= 0.0030) with AA/P response/resistance at 12 weeks. Although statistical analysis showed the significant CNV change at AR locus between baseline and 12-week post-AA/P treatment (p=0.0002), the CNV change was not significantly different between responders and nonresponders (p=0.1697). Conclusions: cfDNA CNV may serve as a sensitive biomarker for prediction of treatment response to AA/P. Further detailed CNV and clinical association analyses are needed to fully validate the potential clinical utilities of cfDNA-based liquid biopsy for clinical management. Citation Format: Meijun Du, Manish Kohli, Chiang-Ching Huang, Liang Wang. Genomic alterations in plasma cell-free DNA predict treatment response to abiraterone acetate and prednisone and survival in metastatic castration-resistant prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1077.

Introduction: Survival in mCRPC stage is heterogeneous and in this stage no molecular predictors of survival are known. Elevated circulating tumor cell (CTC) counts (>5/7.5 ml blood CellSearch® assay) is the only FDA cleared prognostic biomarker in mCRPC stage that predicts poor prognosis but is not routinely performed in clinical practice. We evaluated a novel plasma cfDNA based molecular algorithmic score which is based on copy number variations (CNVs) in multiple candidate genes as a prognostic predictor of survival and also compared it to CTCs. Methods: We obtained biospecimens in a prospective cohort study (NCT#01953640) conducted in mCRPC patients who demonstrated progression on androgen deprivation therapy and were candidates to initiate pre-chemotherapy abiraterone acetate/prednisone (AA/P). Biospecimen collection was performed prior to initiating AA/P and included plasma and CTCs, both obtained at the same time. All samples underwent uniform processing. Cellsearch® assay was used for enumerating CTC counts. Plasma cfDNA was extracted using Qiagen whole blood kit and low pass sequencing was performed in the extracted plasma cfDNA and germline DNA. An algorithmic score based on CNVs in 13 candidate genes was developed after calculating an Absolute Log2 Ratio (ALR) for each pre-selected gene. The sum of ALRs for these 13 genes were combined to make a “Plasma Genome Abnormality” (PGA) score which was evaluated for association with the primary endpoint of overall survival (OS) using Hazard ratio (HR) with significance set at p ≤ 0.05. Association of concurrently collected CTC counts with OS was also determined. The PGA score based survival was adjusted for CTC counts in the final analysis. Results: Plasma and CTCs were available for analysis in 72/90 enrolled patients between 05/2013 and 09/2015 with the last enrolled patient having completed 23.5 months follow-up. 45/72 patients have died of cancer progression and the median OS of the cohort is 26.5 months (IQR: 19-46). The median plasma cfDNA amount obtained was 7.2 (Range: 1.6-70.4)ng/ml plasma and the median CTC count is 1/7.5ml blood (range: 0-308). The median PGA score was 1.108 (Range: 0.56-6.96) and the CNV based PGA score was observed to be associated with OS (HR: 1.63; 95% C.I: 1.144, 2.308); p-value= 0.0066). Association of elevated CTC counts (≥ 5/7.5 ml blood) with OS showed a statistically significant trend (HR: 1.92; CI: 0.99 - 3.7; p-value 0.051). After adjusting for CTC counts, an increased PGA score remained predictive of poor survival (HR: 1.52; 95% CI: (1.04, 2.21); p-value 0.028). Conclusions: In this cohort the PGA score, a molecular algorithm predictor based on plasma cfDNA copy number variations in candidate genes was a better prognostic marker of survival than CTC counts and potentially offers a biomarker tool which could be adopted for wider clinical application. Citation Format: Manish Kohli, Meijun Du, Liang Wang, Chiang-Ching Huang. Prognostic association of plasma cell free DNA (cfDNA) copy number variation based algorithmic score with survival in metastatic castration resistant prostate cancer (mCRPC) [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 4588.

Genome-wide association studies have identified more than 100 common single nucleotide polymorphisms (SNPs) that are associated with prostate cancer risk. However, the vast majority of these SNPs lie in noncoding regions of the genome. To test whether these risk SNPs regulate their target genes through long-range chromatin interactions, we applied capture-based 3C sequencing technology to investigate possible cis-interactions at ten prostate cancer risk loci in six cell lines. We identified significant physical interactions between risk regions and their potential target genes including CAPG at 2p11.2, C2orf43 at 2p24.1, RFX6 at 6q22.1, NFASC at 1q32.1, MYC at 8q24.1 and AGAP7P at 10q11.23. Most of the interaction peaks were co-localized to regions of active histone modification and transcription factor binding sites. Expression quantitative trait locus (eQTL) analysis showed suggestive eQTL signals at rs1446669, rs699664 and rs1078004 for CAPG (p C2orf43 (p = 2.25E-27), rs10993994 and rs4631830 for AGAP7P (p

Objectives Cell free tumor DNA (cfDNA) circulating in blood has a great potential as biomarker for cancer clinical management. The objective of this study is to evaluate if cfDNA in blood plasma is detectable in early stage lung cancer patients. Materials and methods We extracted cfDNAs and tumor tissue DNAs from 8 lung adenocarcinoma patients. We also extracted cfDNAs from 8 normal controls. To evaluate copy number variations (CNV) and identify potential mutations, we performed low pass whole genome sequencing and targeted sequencing of 50 cancer genes. To accurately reflect the tumor-associated genomic abnormality burden in plasma, we developed a new scoring algorithm, plasma genomic abnormality (PGA) score, by summarizing absolute log2 ratios in most variable genomic regions. We performed digital PCR and allele-specific PCR to validate mutations detected by targeted sequencing. Results and conclusions The median yield of cfDNA in 400ul plasma was 4.9ng (range 2.25–26.98ng) in patients and 2.32ng (range 1.30–2.81ng) in controls ( p =0.003). The whole genome sequencing generated approximately 20million mappable sequence reads per subject and 5303 read counts per 1Mb genomic region. Log2 ratio-based CNV analysis showed significant chromosomal abnormality in cancer tissue DNAs and subtle but detectable differences in cfDNAs between patients and controls. Genomic abnormality analysis showed that median PGA score was 9.28 (7.38–11.08) in the 8 controls and 19.50 (5.89–64.47) in the 8 patients ( p =0.01). Targeted deep sequencing in tumor tissues derived from the 8 patients identified 14 mutations in 12 different genes. The PCR-based assay confirmed 3 of 6 selected mutations in cfDNAs. These results demonstrated that the PGA score and cfDNA mutational analysis could be useful tool for the early detection of lung cancer. These blood-based genomic and genetic assays are noninvasive and may sensitively distinguish early stage disease when combined with other existing screening strategies including low-dose CT scanning.

Background: eccDNA (extrachromosomal circular DNA) has been reported in most eukaryotes, including in human cells. However, little is known about the eccDNA profiles in human circulating system such as blood. Method: We extracted plasma cell free DNA (cfDNA) from three patients with advanced stage cancers. For each patient, we tested 10ng, 1ng and 0.1ng cfDNA, each with a technical replicate. The cfDNA samples were subjected to ATP-dependent DNase digestion and whole genome amplification (WGA). An additional 2ng cfDNA from each patient skipped the DNase digestion and went directly to WGA step. We sequenced the amplified DNAs using the Illumina 100bp PE read protocol. We analyzed sequencing data using a custom split reads-based strategy to retrieve circular DNA molecules. We further analyzed eccDNAs for their biofeatures using functional annotation tools. Results: We received sequence reads at ~14.8 million (11.4~22.4) for each sample. When read depth≥ 3 as a cutoff, we detected a total number of 2542, 1578, and 57 unique eccDNAs in three patients while negative controls showed 500bp, 41%) (p Conclusion: Human plasma contains an abundant number of eccDNAs. Distribution of these eccDNAs is nonrandom event and is likely enriched in genomic regions with known functional consequences and genes that play a role in gene regulations. Further characterization of circulating eccDNAs in peripheral blood will facilitate understanding of their molecular mechanisms and potential clinical utilities. Citation Format: Jing Zhu, Meijun Du, Peng Zhang, Liang Wang. Detection and characterization of extrachromosomal circular DNA in human plasma [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 4244. doi:10.1158/1538-7445.AM2017-4244

Background: Circulating cell-free DNA (cfDNA) analysis is emerging as a less invasive approach to assessing tumor genomic alterations in cancer patients. Although high concordance has been reported between tumor tissue NGS and cfDNA in studies investigating specific genetic alterations, the fidelity of cfDNA to tumor tissue DNA in the global genomic scale is largely unknown. In a correlative study of a prospective clinical trial (NCT# 01953640) conducted in mCRPC stage patients treated with abiraterone acetate/prednisone (A/P), we evaluated correlation of genomic CNA in tumor DNA obtained from biopsy of metastatic lesions and matched plasma cfDNA. Methods: mCRPC patients (pts) underwent two image-guided core biopsies of metastases prior to initiation of A/P (visit 1, V1) and 12 weeks after treatment (visit 2, V2). At both time points plasma was obtained at the time of the core biopsies and cfDNA was extracted using DNA Blood Mini Kit (Qiagen, Valencia, CA). High coverage (for tumor tissue) and low coverage (for cfDNA) whole genome sequencing reads were first mapped to the human genome hg19. Read counts (RC) from the mapped sequence files were then binned into 1Mb windows. The RC ratio in each genomic bin was calculated by comparing tumor tissue DNA to lymphocyte gDNA derived from the same patient, and was further log2 transformed, corrected for GC content, and normalized by CGHnormaliter. The fully normalized log2 ratios data was subjected to segmentation using DNAcopy algorithm. Results: Between 05/2013 and 09/2015, 92 patients (pts) were enrolled of which tissue and plasma NGS data both visits was available for 18 pts. The correlation of CNAs between tumor tissue and its cfDNA counterpart ranges from 0.013 to 0.83 for V1 samples, and -0.05 to 0.9 for V2 samples. The decreased correlation in some pairs of samples is largely due to low tumor content and heterogeneity in the cfDNA. Although there is a wide range of correlation, commonly shared CNAs were identified in multiple chromosomal regions, including loss in 8p, gain in 8q and chromosome 5 and X. On the other hand, several genomic regions show inconsistent CNAs between tumor tissue and cfDNA among 15 pairs of samples in which cfDNA have large tumor content. These include: loss in 6q, 1p, 2p, 9q, 11q, 13p, 15p, 20p and gain in 8q and 10p in cfDNA but not in tumor tissue; marked loss in chromosome 22 and gain in p arm of X chromosome in tumor tissue but much less evident in cfDNA. Conclusion: High concordance of CNAs between cfDNA and tumor tissue DNA can be achieved given sufficient tumor content of cfDNA. However, more CNAs can be identified in cfDNA than its tumor tissue counterpart. Our result suggests that cfDNA NGS is a useful tool to investigate clonal evolution associated with cancer progression. Citation Format: Chiang-Ching Huang, Meijun Du, Liguo Wang, Yen-chen Lin, Hua Huang, Liewei Wang, Liang Wang, Manish Kohli. Comparison of copy number aberrations (CNAs) between plasma cell free DNA (cfDNA) and tissue DNA in metastatic castrate resistant prostate cancer (mCRPC) [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 2733. doi:10.1158/1538-7445.AM2017-2733

Background Small cell lung cancer (SCLC) is a highly aggressive malignancy with rapid development of chemo resistance and a high rate of recurrence. Although tumor tissues-based studies have shown characteristic genomic changes in SCLC, it is little known about tumor-derived genomic variations in cell free DNA (cfDNA), a promising source of genetic materials for monitoring disease progression, evaluating treatment efficacy and predicting clinical outcomes. Methods We collected plasma from 26 SCLC patients and extracted cfDNAs from 250μl of plasma. We prepared DNA libraries and analyzed sequencing data using a various bioinformatic tools. We binned read counts from the mapped sequence files into 60kb windows and performed log2 ratio-based copy number analysis using segmentation-based approach. We compared copy number changes detected in cfDNA to previously reported genomic regions from tumor tissues-based studies. Results The whole genome sequencing generated approximately 9.4 million mappable sequence reads per subject and 175 read counts per 60kb genomic region. Copy number analysis using log2 ratios between cfDNA and matched gDNA showed significant genomic abnormalities in 17 of 26 plasma samples. Overall, we observed the most frequent deletion of 3p, 13q,17p,10p and amplification of 3q, 5p, 8q,1p, which were consistent with previous reports from tumor tissues-based studies. To further define the copy number changes, we performed a detailed analysis at chromosomal regions showing frequent aberrations in SCLC. Among these, deletion of gene RASSF1 at 3p21.3 was most frequently observed (12 deletions in 26 samples). Another common deletion was RB1 at 13q14 (5/26), TP53 at 17p13 (4/26). The most common amplification was involved in MYC gene at 8q24(18/26). Other common amplifications included SOX2 at 3q26 (17/26), MYCL at 1p34.2 (9/26) and FGF10 at 5p13 (9/26). We also observed low frequent genomic alterations at other genomic regions such as PTEN loss at 10q23(1/26) and FHIT loss at 3p14 (1/26). Interestingly, we observed contradictory genomic changes at MAD1L1 locus. Among the 26 cfDNA samples, we detected ten patients with MAD1L1 loss and three patients with MAD1L1 gain. Past studies have shown that the MAD1L1 locus at 7p22 is frequently ampliated in SCLC patients. We are currently analyzing if these genomic variations are associated with disease progression and overall survival. Conclusions This study provides a comprehensive analysis of genomic alterations in plasma cfDNAs from SCLC. We identified multiple candidate genes whose genomic aberrations may be used as biomarkers for accessing treatment efficacy and predicting prognosis. Our result supports blood plasma as an alternative sample source for tumor-associated genomic analysis in SCLC patients. Further targeted sequencing and clinical association analyses is needed to fully validate the potential clinical utilities of cfDNA-based liquid biopsy for clinical management of this deadly disease. Citation Format: Meijun Du, Hannah Fisher, Peng Zhang, Huijuan Zhang, Liang Wang. Genomic variations in plasma cell free DNA from small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5395. doi:10.1158/1538-7445.AM2017-5395

Background. By SNP genotyping and RNA sequencing of 471 normal prostate samples, we recently created a prostate tissue-based eQTL dataset and identified significant eQTL signals at 51 prostate cancer risk loci. To functionally characterize these risk SNPs, we developed a massively parallel sequencing technology to screen SNPs for their allele-dependent protein binding differences. We combined this technology (called SNPs-seq) with another high throughput assay (called STARR-seq) to screen the risk loci with significant prostate-specific eQTL signals. Methods. To select candidate functional SNPs in eQTL regions, we took advantage of existing epigenomic datasets and available tools including ENCODE, HaploReg, and Regulome. For all selected SNPs, we first made allele-specific double-strand oligos and performed DNA-protein binding assays. We then performed sequencing analysis on the protein-bound DNA oligos and determined allele-specific protein binding differences. To evaluate reproducibility of SNPs-seq, we performed each assay in duplicates. We cloned SNPs-seq screened SNP regions showing allele-specific protein binding differences into the STARR-seq vector to further determine allele-specific enhancer activities. Finally, we performed EMSA and luciferase reporter assays to validate a set of promising candidate SNPs. Results. From 51 risk loci with strong eQTL signals, we selected 374 SNPs with strong indication of regulatory potential, as evidenced by overlapping with epigenomic marks. When comparing technical duplicates, sequence read counts from the SNPs-seq showed significant correlation with r2>=0.99. By normalizing input controls, we found 101 of the 374 SNPs showing significant allelic protein binding differences (>=1.5-fold binding difference between variant and reference alleles). Interestingly, three published functional SNPs (rs12769019, rs10993994, and rs4907792) were also among the significant SNPs, validating SNPs-seq as functional SNP screening tool. To further validate the candidate SNPs from SNPs-seq, we applied STARR-seq and tested the 101 SNPs-containing sequences (371-686bp) in LNCaP cell line under androgen treatment. This analysis revealed 11 SNPs that not only demonstrated enhancer/repressor activity but also functioned with allelic differences. EMSA and luciferase reporter assays confirmed 6 SNPs with allele-dependent enhancer/repressor activity. Conclusions. We developed a high throughput sequencing-based technology to screen large number candidate SNPs for their allelic protein binding differences. The SNPs-seq coupled with STARR-seq will provide a powerful strategy for functionally characterizing risk loci in prostate cancer and other common diseases. Further understanding genetic role of prostate cancer etiology may facilitate the translation of population-based discovery into biological mechanisms and eventually benefit clinical practice. Citation Format: Peng Zhang, Jing Zhu, Sufyan Suleman, Yong-Chen Guo, Mei-Jun Du, Li-Dong Wang, Gong-Hong Wei, Liang Wang. Functional characterization of prostate cancer risk loci by SNPs-seq and STARR-seq [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1280. doi:10.1158/1538-7445.AM2017-1280

152 Background: Plasma cfDNA ARcopy number variations (CNV) have been suggested to have prognostic significance in mCRPC stage. We compared plasma AR amplification ( ARamp) with CTC counts (the only FDA approved prognostic marker in mCRPC stage) in a prospective trial of patients (pts) progressing on androgen deprivation therapy. Methods: Plasma and CTC counts were obtained concurrently as part of a prospective clinical trial in mCRPC stage pts initiating abiraterone acetate (NCT# 01953640). Plasma cfDNA was purified using DNA Blood Mini Kit (Qiagen, Valencia, CA) and AR CNVs were detected using QuantStudio3D digital PCR system (Life Technologies, Carlsbad, CA, USA). CTC enumeration was performed using the FDA cleared CellSearch platform. The distributions of overall survival (OS) time based on ARamp status and CTC counts ( < 5 vs ≥ 5 cells) were estimated using the Kaplan-Meier method. Log-rank tests were used to test association of ARamp status and CTC counts with overall survival (OS). Univariate and multivariate logistic regression models and ROC evaluated association of OS at 24 months by ARamp status and CTCs ≥ 5. Results: Between 05/2013 and 09/2015, 92 pts were enrolled of which complete CTC, plasma and outcome data was available for 70 pts. Median age of the cohort (range) was 72 yrs (39-91); Median PSA–16.2 ng/ml (range: 0.9-2026.0); Median CTC count was 2.0 (range: 0-372); 58/70 pts had bone metastases; 38/70 had high volume and 32/70 had low volume metastatic disease at the time of sample collection. The median (range) follow up time at the time of this analysis was: 26.5 (7.9-37.0) months at which time 28/70 patients had died. ARamp was present in 19/70 pts while 51/70 did not have amplification. 24/70 had CTCs ≥ 5. A significant association of increased ARamp status with OS (p < 0.0001, log-rank test; AUC: 0.66) as well as with CTC counts ≥ 5 (p = 0.001, log-rank test; AUC: 0.67) was observed. Combining CTC status and plasma ARamp status the AUC was 0.75. Conclusions: Plasma cfDNA ARamp status and CTC counts have comparable and clinically meaningful prognostic marker value in mCRPC stage patients.

Extracellular microRNAs (miRNAs) embedded in circulating exosomes may serves as prognostic biomarkers in cancer.To identify and evaluate plasma exosomal miRNAs for prognosis in castration-resistant prostate cancer (CRPC).RNA sequencing was performed to identify candidate exosomal miRNAs associated with overall survival in a screening cohort of 23 CRPC patients. Candidate miRNAs were further evaluated for prognosis using quantitative real-time polymerase chain reaction in a follow-up cohort of 100 CRPC patients.Cox regression and Kaplan-Meier survival analyses were used to evaluate survival association using candidate miRNAs along with clinical prognostic factors.RNA sequencing in screening cohort generated approximately 6.80 million mappable reads per patient. Of those with normalized read counts ≥ 5, 43% were mapped to miRNAs for a total of 375 known and 57 novel miRNAs. Cox regression analysis identified an association of miR-1290, -1246, and -375 with overall survival (false discover rate0.05). Of those, higher levels of miR-1290 and -375 were significantly associated with poor overall survival (p0.004) in the follow-up cohort. Incorporation of miR-1290/-375 into putative clinical prognostic factors-based models in CRPC stage significantly improved predictive performance with a time-dependent area under the curve increase from 0.66 to 0.73 (p = 6.57 × 10(-6)).Plasma exosomal miR-1290 and miR-375 are promising prognostic biomarkers for CRPC patients. Prospective validation is needed for further evaluation of these candidate miRNAs.In this study, we evaluated whether small RNAs circulating in blood could be used to predict clinical outcomes in late-stage prostate cancer patients. We identified two blood-based small RNAs whose levels showed significant association with survival. Our results warrant further investigation because the noninvasive blood-based test has great potential in the management of late-stage prostate cancer.

Background: Castration-resistant prostate cancer (CRPC) patients are commonly treated with the chemotherapy drug, docetaxel. We previously reported that elevated miR-375 was significantly associated with poor overall survival of CRPC patients. However, the biological role and mechanism of action of miR-375 in CRPC chemotherapy remain unknown. In this study, we evaluated if miR-375 induced chemo-resistance through regulating target genes associated with multiple drug resistance. Methods: We first compared miR-375 expression level between prostate cancer tissues and normal prostate tissues using TCGA prostate cancer data. To examine the role of miR-375 in docetaxel sensitivity, we then transfected miR-375 using a pre-miRNA lentiviral vector and examined the effect of the exogenously overexpressed miR-375 on cell growth in two prostate cancer cell lines, DU145 and PC3. To determine the effect of overexpressed miR-375 on tumor growth and chemo-resistance in vivo, we subcutaneously injected cells with miR-375 overexpression into mice. Lastly, we utilized qRT-PCR and western blot assay to examine two miR-375 target genes, SEC23A and YAP1, for their expression changes after miR-375 transfection. Results: By comparing 495 tumor tissues and 52 normal tissues, we found that miR-375 was significantly overexpressed in prostate cancer tissues (8.45 fold increase, p value = 1.98E-23). Docetaxel treatment induced higher expression of miR-375 with 5.83 and 3.02 fold increases in DU145 and PC3, respectively. Interestingly, miR-375 appeared to play a contradictory role in determining prostate cancer fate. We found that miR-375 overexpression caused cell proliferation inhibition, cell cycle arrest and cell apoptosis. However, we also found that the elevated miR-375 significantly reduced cell sensitivity to docetaxel treatment in vitro, evidenced by less apoptotic cells and higher cell proliferation. Mouse models also showed that the increased miR-375 expression was resistant to docetaxel treatment, demonstrated by greater tumor weight and sizes in miR-375 transfected cells than in empty vector controls. In addition, previous studies in other laboratories have shown that SEC23A and YAP1 are miR-375 target genes and are able to reduce cancer cell growth. We therefore examined expression levels of the two genes and observed significant expression reduction of both proteins and mRNAs in the miR-375 transfected prostate cancer cell lines. TCGA dataset analysis further confirmed the negative correlations between miR-375 and the two target genes (r = -0.62 and -0.56 for SEC23A and YAP1, respectively; p Conclusions: miR-375 may confer chemo-resistance to docetaxel treatment during prostate cancer chemotherapy through directly targeting SEC23A and YAP1. Our study implies that miR-375 or its target genes, SEC23A or YAP1 might serve as potential diagnostic and/or therapeutic targets to overcome chemo-resistance in CRPC treatment. Citation Format: Yuan Wang, Rachel Lieberman, Jing Pan, Qi Zhang, Meijun Du, Ming You, Liang Wang. miR-375 induces docetaxel resistance in prostate cancer by targeting SEC23A and YAP1. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4948.

Androgen deprivation therapy (ADT) for hormone-sensitive prostate cancer (HSPC) and chemotherapy for castration-resistant prostate cancer (CRPC) are considered standard of care. However, there are no clinical tests or factors that can reliably predict treatment responses for the advanced prostate cancer patients. Examination of tumor component in circulation, also known as liquid biopsy, has shown promise for predicting treatment outcomes and survival. To capture the tumor-derived genomic alterations and decrease the surrogacy of using PSA measurements as is currently performed, we propose a Plasma Genomic Abnormality (PGA) score and Treatment Efficacy (TEff) index based on copy number variations as estimated by cell free DNA (cfDNA) in plasma. We first performed whole genome and targeted sequencing (NimbleGen comprehensive cancer panel) to examine plasma cfDNA for tumor-associated genetic and genomic aberrations in 10 HSPC patients receiving ADT and 10 CRPC patients receiving chemotherapy. For each patient, we tested plasma from two time points: pre-treatment and 4 months post-treatment. We then calculated the PGA score by summing the most significant genomic abnormalities with higher PGA score indicating greater tumor DNA content in cfDNA. TEff index was derived from comparison of PGA score difference between treatments with higher TEff index reflecting a better treatment response. The sequencing-based copy number analysis revealed locus-specific gains or losses including those previously reported in prostate cancers, such as 8q gains, AR amplifications, PTEN losses and TMPRSS2-ERG fusions. We found that overall PGA score was significantly higher in CRPC patients than in HSPC patients (p Citation Format: Liang Wang, Shu Xia, Meijun Du, Rachel Ditmar, Tiezheng Yuan, Yongchen Guo, Yuan Wang, Adam Lee, Michael Tschannen, Elizabeth Worthey, Howard Jacobs, Chiang-Ching Huang, Manish Kohli. Plasma genetic and genomic abnormalities predict treatment response and clinical outcome in advanced prostate cancer. [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 5231. doi:10.1158/1538-7445.AM2015-5231

PURPOSE: Cell free circulating microvesicles (in particular, exosomes) contain proteins and nuclei acids that may serve as biomarkers for disease diagnosis and prognosis. The goal of this study was to identify exosomal microRNAs that may predict clinical outcome of patients with castrate resistant prostate cancer (CRPC). EXPERIMENTAL DESIGN: In discovery stage, we performed RNA sequencing using plasma exosomal RNAs derived from 36 patients with CRPC. We mapped the sequence reads to known miRbase (Release 19, 2043 entries) and normalized the miRNA abundance by sequence counts per million mappable reads. We applied Cox regression analysis to identify the miRNAs that were associated with overall survival of CRPC. In validation stage, we first examined an exosomal small RNA sequencing dataset consisting of 192 individuals with various health conditions. We used Normfinder and Bestkeeper to select most stably expressed miRNAs as candidates for normalization references. We then applied real-time qRT-PCR assays to test selected candidate reference miRNAs and survival-related miRNAs in additional 100 CRPC patients. From validated miRNAs, we constructed multivariate models to predict overall survival for this group of patients. RESULTS RNA sequencing generated over 6 million mappable reads per patient in the initial discovery cohort. Among these reads, ∼45% were mapped to known miRNAs for a total of 483 known and 275 novel miRNAs with normalized read counts ≥ 5. The median follow-up time for the 36 CRPC patients was 35.6 months during which 10 patients had died. Cox regression analysis identified four microRNAs (miR-1290, -1246, -375 and a predicted miRNA at chromosome 12) that were associated with overall survival (FDR CONCLUSIONS: Plasma exosomal miRNAs provide an easily accessible resource for biomarker development in prognosis of advanced prostate cancer. Exosomal miR-1290 and miR-375 are associated with overall survival in CRPC patients. miR-30a/e-5p, especially the geometric mean of miR-30a/e-5p, are qualified endogenous references for real-time qPCR. Further confirmation of these findings is needed for prognostic and predictive biomarker in development of CRPC stage. Citation Format: Xiaoyi Huang, Tiezheng Yuan, Meihua Liang, Meijun Du, Shu Xia, Rachel Louise Dittmar, Zhifu Sun, Yan Lu, Stephen N. Thibodeau, Lisa Boardman, Manish Kohli, Liang Wang. Exosomal miR-1290 and miR-375 as prognostic markers in metastatic castrate resistant prostate cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 574. doi:10.1158/1538-7445.AM2014-574