Du, Meijun

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

Abstract 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.

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 ≥ 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–Meier analysis showed overall survival’s association with the amplification of AR-En (p = 0.02, HR = 1.68 (1.07–2.65)), AR-E8 (p = 0.02, HR = 1.78 (1.08–2.92)) and AR-En-E8 (the combination of AR-En and AR-E8 (p = 0.009, HR = 1.77 (1.15–2.73)). 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.

Abstract 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.

Abstract 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.

Abstract 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

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.

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.