Dittmar, Rachel L.

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.

Abstract 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&lt;0.05). Of six selected candidate miRNAs, miR-30a/e-5p performed the best in their stability as endogenous references for real-time qRT-PCR. When normalized by these internal controls, we confirmed two of the three known miRs (miR-1290 and -375) showing significant association with overall survival in the validation cohort (p&lt;0.001) with a median follow up time of 12.5 (range: 1-41) months at which time 28 patients had died. The median overall survival for patients with high (N=21) and low (N=40) expression in both miR-1290 and -375 was 7.2 (1-26.7) months and 15.3 (1-41) months, respectively (p&lt;0.05). The miR-1290/-375-based prediction model consistently showed better predictive performance with AUC=72% than clinical variable-based model with AUC=64%. 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