Search

Supplementary Table #1

Supplemental Figure 1: Biodistribution data reflecting the accumulation of 68Ga-citrate over time in the tissues of male nu/nu mice bearing PC3 xenografts. Peak radiotracer uptake was observed at 4 hours post injection. Supplemental Figure 2: Biodistribution data reflecting the accumulation of 68Ga-citrate at 4 hours post injection in the tissues of male nu/nu mice bearing 22Rv1 xenografts. Supplemental Figure 3: Biodistribution data collected 4 hours post injection of 68Ga-citrate in male nu/nu mice bearing subcutaneous PC3 xenografts, and 48 hours post injection of 89Zr-Tf in male nu/nu mice with subcutaneous PC3 xenografts. Supplemental Figure 4. BET inhibitors suppress MYC and transferrin uptake in MYC positive prostate cancer cell lines. Supplemental Figure 5: Biodistribution data collected 4 hours post injection of 68Ga-Tf in male nu/nu mice bearing subcutaneous PC3 or 22Rv1 xenografts. Mice were treated with vehicle, (+)-JQ1 (25 mg/kg, BID), or iBET (50 mg/kg, daily oral gavage) for 5 days prior to radiotracer injection. Supplemental Figure 6: Representative PET data collected 4 hours post injection of 68Ga-citrate or 48 hours post injection of 89Zr-Tf in male nu/nu mice bearing subcutaneous 22Rv1 xenografts. Supplemental Figure 7: Paired PET imaging plus CT-guided biopsy of tracer-avid liver metastasis in patient-003 with mCRPC. Supplemental Figure 8: H&E staining of the PDX tissue isolated from the liver metastasis of patient-003 shows morphological features consistent with the liver biopsy. Supplemental Figure 9. Biodistribution data acquired 4 hours post injection of 68Ga-citrate shows the accumulation of the radiotracer in the PDX tissue and selected normal mouse tissues. Supplemental Table 1. A summary of the tumor:blood and tumor: muscle ratios for 68Ga-citrate Supplemental Table 2. A summary of the patient characteristics in this study. Supplemental Table 3. A summary of the test/re-test data for 68Ga-citrate in normal tissues. Data are presented as SUVmax. Supplementary Table 4. A summary of the histological subclassifications for the prostate cancer biopsies acquired during this study.

Early detection of pancreatic ductal adenocarcinoma (PDAC) is key to improving patient outcomes; however, PDAC is usually diagnosed late. Therefore, blood-based minimally invasive biomarker assays for limited volume clinical samples are urgently needed. A novel miRNA profiling platform (Abcam Fireplex-Oncology Panel) was used to investigate the feasibility of developing early detection miRNA biomarkers with 20 μL plasma from a training set (58 stage II PDAC cases and 30 controls) and two validation sets (34 stage II PDAC cases and 25 controls; 44 stage II PDAC cases and 18 controls). miR-34a-5p [AUC = 0.77; 95% confidence interval (CI), 0.66–0.87], miR-130a-3p (AUC = 0.74; 95% CI, 0.63–0.84), and miR-222-3p (AUC = 0.70; 95% CI, 0.58–0.81) were identified as significantly differentially abundant in plasma from stage II PDAC versus controls. Although none of the miRNAs individually outperformed the currently used serologic biomarker for PDAC, carbohydrate antigen 19-9 (CA19-9), combining the miRNAs with CA 19-9 improved AUCs from 0.89 (95% CI, 0.81–0.95) for CA 19-9 alone to 0.92 (95% CI, 0.86–0.97), 0.94 (95% CI, 0.89–0.98), and 0.92 (95% CI, 0.87–0.97), respectively. Gene set enrichment analyses of transcripts correlated with high and low expression of the three miRNAs in The Cancer Genome Atlas PDAC sample set. These miRNA biomarkers, assayed in limited volume plasma together with CA19-9, discriminate stage II PDAC from controls with good sensitivity and specificity. Unbiased profiling of larger cohorts should help develop an informative early detection biomarker assay for diagnostic settings.Prevention Relevance:Development of minimally invasive biomarker assays for detection of premalignant disease and early-stage pancreatic cancer is key to improving patient survival. This study describes a limited volume plasma miRNA biomarker assay that can detect early-stage resectable pancreatic cancer in clinical samples necessary for effective prevention and clinical intervention.

Noninvasive biomarkers that detect the activity of important oncogenic drivers could significantly improve cancer diagnosis and management of treatment. The goal of this study was to determine whether 68Ga-citrate (which avidly binds to circulating transferrin) can detect MYC-positive prostate cancer tumors, as the transferrin receptor is a direct MYC target gene. PET imaging paired with 68Ga-citrate and molecular analysis of preclinical models, human cell-free DNA (cfDNA), and clinical biopsies were conducted to determine whether 68Ga-citrate can detect MYC-positive prostate cancer. Importantly, 68Ga-citrate detected human prostate cancer models in a MYC-dependent fashion. In patients with castration-resistant prostate cancer, analysis of cfDNA revealed that all patients with 68Ga-citrate avid tumors had a gain of at least one MYC copy number. Moreover, biopsy of two PET avid metastases showed molecular or histologic features characteristic of MYC hyperactivity. These data demonstrate that 68Ga-citrate targets prostate cancer tumors with MYC hyperactivity. A larger prospective study is ongoing to demonstrate the specificity of 68Ga-citrate for tumors with hyperactive MYC.Implications: Noninvasive measurement of MYC activity with quantitative imaging modalities could substantially increase our understanding of the role of MYC signaling in clinical settings for which invasive techniques are challenging to implement or do not characterize the biology of all tumors in a patient. Moreover, measuring MYC activity noninvasively opens the opportunity to study changes in MYC signaling in patients under targeted therapeutic conditions thought to indirectly inhibit MYC. Mol Cancer Res; 15(9); 1221–9. ©2017 AACR.

Supplemental Figure #1

Purpose: Ribociclib, a CDK4/6 inhibitor, demonstrates preclinical antitumor activity in combination with taxanes. We evaluated the safety and efficacy of ribociclib plus docetaxel in a phase Ib/II study in metastatic castration-resistant prostate cancer (mCRPC). Patients and Methods: Patients had chemotherapy-naïve mCRPC with progression on ≥ 1 androgen receptor signaling inhibitor (ARSI). The phase II primary endpoint was 6-month radiographic progression-free survival (rPFS) rate, with an alternative hypothesis of 55% versus 35% historical control. Circulating tumor cells (CTC) were collected at baseline and genomically profiled. Result: Forty-three patients were enrolled (N = 30 in phase II). Two dose-limiting toxicities were observed (grade 4 neutropenia and febrile neutropenia). The recommended phase II dose (RP2D) and schedule was docetaxel 60 mg/m2 every 21 days plus ribociclib 400 mg/day on days 1–4 and 8–15 with filgrastim on days 5–7. At the RP2D, neutropenia was the most common grade ≥ 3 adverse event (37%); however, no cases of febrile neutropenia were observed. The primary endpoint was met; the 6-month rPFS rate was 65.8% [95% confidence interval (CI): 50.6%–85.5%; P = 0.005] and median rPFS was 8.1 months (95% CI, 6.0–10.0 months). Thirty-two percent of evaluable patients achieved a PSA50 response. Nonamplified MYC in baseline CTCs was associated with longer rPFS (P = 0.052). Conclusions: The combination of intermittent ribociclib plus every-3-weeks docetaxel demonstrated acceptable toxicity and encouraging efficacy in ARSI-pretreated mCRPC. Genomic profiling of CTCs may enrich for those most likely to derive benefit. Further evaluation in a randomized clinical trial is warranted.

Gene fusions occurring in one or more of the 24 tumors.

Supplementary Figure from A Phase Ib/II Study of the CDK4/6 Inhibitor Ribociclib in Combination with Docetaxel plus Prednisone in Metastatic Castration-Resistant Prostate Cancer

Purpose: The three main treatment options for primary prostate cancer are surgery, radiation, and active surveillance. Surgical and radiation intervention for prostate cancer can be associated with significant morbidity. Therefore, accurate stratification predictive of outcome for prostate cancer patients is essential for appropriate treatment decisions. Nomograms that use clinical and pathologic variables are often used for risk prediction. Favorable outcomes exist even among men classified by nomograms as being at high risk of recurrence.Experimental Design: Previously, we identified a set of DNA-based biomarkers termed Genomic Evaluators of Metastatic Prostate Cancer (GEMCaP) and have shown that they can predict risk of recurrence with 80% accuracy. Here, we examined the risk prediction ability of GEMCaP in a high-risk cohort and compared it to a Kattan nomogram.Results: We determined that the GEMCaP genotype alone is comparable with the nomogram, and that for a subset of cases with negative lymph nodes improves upon it.Conclusion: Thus, GEMCaP shows promise for predicting unfavorable outcomes for negative lymph node high-risk cases, where the nomogram falls short, and suggests that addition of GEMCaP to nomograms may be warranted. Clin Cancer Res; 16(1); 195–202

Allele frequencies for 128 ancestry informative single nucleotide polymorphisms, with correlations (r values) between Structure model estimates and actual allele frequencies for our 24 African American (AA) patients, plus 146 other AA and 204 European American (EA) samples ; Individual patient admixture proportions from three ancestral populations ; Somatic transition and transversion rates and ratios in our data compared to the TCGA data ; Heatmap showing log2-ratios of tumor versus normal copy number in 100Kb segments across the genome ; Circos plots showing rearrangementsb and copy numberc in the 24 tumor samples, by ETS fusion status .

Supplementary Table from A Phase Ib/II Study of the CDK4/6 Inhibitor Ribociclib in Combination with Docetaxel plus Prednisone in Metastatic Castration-Resistant Prostate Cancer

PDF - 99K, Detailed methods.

Supplementary Data from A Group of Genome-Based Biomarkers That Add to a Kattan Nomogram for Predicting Progression in Men with High-Risk Prostate Cancer

Prostate cancer is the most frequently diagnosed and second most fatal nonskin cancer among men in the United States. African American men are two times more likely to develop and die of prostate cancer compared with men of other ancestries. Previous whole genome or exome tumor-sequencing studies of prostate cancer have primarily focused on men of European ancestry. In this study, we sequenced and characterized somatic mutations in aggressive (Gleason ≥7, stage ≥T2b) prostate tumors from 24 African American patients. We describe the locations and prevalence of small somatic mutations (up to 50 bases in length), copy number aberrations, and structural rearrangements in the tumor genomes compared with patient-matched normal genomes. We observed several mutation patterns consistent with previous studies, such as large copy number aberrations in chromosome 8 and complex rearrangement chains. However, TMPRSS2-ERG gene fusions and PTEN losses occurred in only 21% and 8% of the African American patients, respectively, far less common than in patients of European ancestry. We also identified mutations that appeared specific to or more common in African American patients, including a novel CDC27-OAT gene fusion occurring in 17% of patients. The genomic aberrations reported in this study warrant further investigation of their biologic significant role in the incidence and clinical outcomes of prostate cancer in African Americans. Cancer Res; 76(7); 1860–8. ©2016 AACR.

Evaluation of prostate cancer prognosis after surgery is increasingly relying upon genomic analyses of tumor DNA. We assessed the ability of the biomarker panel Genomic Evaluators of Metastatic Prostate Cancer (GEMCaP) to predict biochemical recurrence in 33 European American and 28 African American prostate cancer cases using genome-wide copy number data from a previous study. “Biomarker positive” was defined as ≥20% of the 38 constituent copy number gain/loss GEMCaP loci affected in a given tumor; based on this threshold, the frequency of a positive biomarker was significantly lower in African Americans (n = 2; 7%) than European Americans (n = 11; 33%; P = 0.013). GEMCaP positivity was associated with risk of recurrence [hazard ratio (HR), 5.92; 95% confidence interval (CI), 2.32–15.11; P = 3 × 10−4] in the full sample and among European Americans (HR, 3.45; 95% CI, 1.13–10.51; P = 0.032) but was not estimable in African Americans due to the low rate of GEMCaP positivity. Overall, the GEMCaP recurrence positive predictive value (PPV) was 85%; in African Americans, PPV was 100%. When we expanded the definition of loss to include copy-neutral loss of heterozygosity (i.e., loss of one allele with concomitant duplication of the other), recurrence PPV was 83% for European American subjects. Under this definition, 5 African American subjects had a positive GEMCaP test value; 4 went on to develop biochemical recurrence (PPV = 80%). Our results suggest that the GEMCaP biomarker set could be an effective predictor for both European American and African American men diagnosed with localized prostate cancer who may benefit from immediate aggressive therapy after radical prostatectomy. Cancer Epidemiol Biomarkers Prev; 23(8); 1677–82. ©2014 AACR.

CCR Translation on the Article from A Group of Genome-Based Biomarkers That Add to a Kattan Nomogram for Predicting Progression in Men with High-Risk Prostate Cancer

Supplementary Figure 1: Distribution of GEMCaP scores* in African American and European American prostate cancer cases. Supplementary Figure 2: Bland-Altman plot assessing the agreement between copy number estimates for the GEMCaP loci for four African American tumor samples profiled using both sequencing and SNP array technologies. Supplementary Table 1: Demographic and clinicopathologic characteristics of the study participants (n=62) by self-identified race-ethnicity. Supplementary Table 2: GEMCaP loci human genome build 19 base-pair locations. Supplementary Table 3: Comparison of copy number estimates from next generation sequencing and SNP array technologies for the GEMCaP loci from four African American prostate tumors. Supplementary Table 4: Evidence for CNAs in other studies of African American prostate tumors for the GEMCaP loci.

Purpose:Ribociclib, a CDK4/6 inhibitor, demonstrates preclinical antitumor activity in combination with taxanes. We evaluated the safety and efficacy of ribociclib plus docetaxel in a phase Ib/II study in metastatic castration-resistant prostate cancer (mCRPC).Patients and Methods:Patients had chemotherapy-naïve mCRPC with progression on ≥ 1 androgen receptor signaling inhibitor (ARSI). The phase II primary endpoint was 6-month radiographic progression-free survival (rPFS) rate, with an alternative hypothesis of 55% versus 35% historical control. Circulating tumor cells (CTC) were collected at baseline and genomically profiled.Result:Forty-three patients were enrolled (N = 30 in phase II). Two dose-limiting toxicities were observed (grade 4 neutropenia and febrile neutropenia). The recommended phase II dose (RP2D) and schedule was docetaxel 60 mg/m2 every 21 days plus ribociclib 400 mg/day on days 1–4 and 8–15 with filgrastim on days 5–7. At the RP2D, neutropenia was the most common grade ≥ 3 adverse event (37%); however, no cases of febrile neutropenia were observed. The primary endpoint was met; the 6-month rPFS rate was 65.8% [95% confidence interval (CI): 50.6%–85.5%; P = 0.005] and median rPFS was 8.1 months (95% CI, 6.0–10.0 months). Thirty-two percent of evaluable patients achieved a PSA50 response. Nonamplified MYC in baseline CTCs was associated with longer rPFS (P = 0.052).Conclusions:The combination of intermittent ribociclib plus every-3-weeks docetaxel demonstrated acceptable toxicity and encouraging efficacy in ARSI-pretreated mCRPC. Genomic profiling of CTCs may enrich for those most likely to derive benefit. Further evaluation in a randomized clinical trial is warranted.

PDF - 159K, Supplementary Figure 1 - Distribution of GEMCaP scores in African American and European American prostate cancer cases. Supplementary Figure 2 - Bland-Altman plot assessing the agreement between copy number estimates for the GEMCaP loci for four African American tumor samples profiled using both sequencing and SNP array technologies. Supplementary Table 1 - Demographic and clinicopathologic characteristics of the study participants (n=62) by self-identified race-ethnicity. Supplementary Table 2 - GEMCaP loci human genome build 19 base-pair locations. Supplementary Table 3 - Comparison of copy number estimates from next generation sequencing and SNP array technologies for the GEMCaP loci from four African American prostate tumors. Supplementary Table 4 - Evidence for CNAs in other studies of African American prostate tumors for the GEMCaP loci.

XLS file - 49K, CpG sites methylated in 100% of CRPC.

XLS file - 15K, Candidate Gene List and Copy number:Expression Correlation for Frequently Aberrant Genes.

XLS file - 32K, Detailed TMPRSS2-ERG and TMPRSS2-ETS2 aCGH and Methylation Mapping.

XLS file - 15K, Genes with copy number aberrations in _>66% of samples (N=15).

PDF file - 7.7K, Hierarchical Clustering of CRPC Samples by Copy Number and Expression: Clustering of samples by: (A) whole genome copy number (x axis = aCGH probes sorted by chromosome, chromosome 1 on left, chromosome 23 on right, green=gain, red=loss). (B) expression level of frequently copy aberrant genes (x axis=gene expression probes red=overexpressed, blue=underexpressed.

Distinguishing indolent from aggressive prostate cancer remains a key challenge for decision making regarding prostate cancer management. A growing number of biomarkers are now available to help address this need, but these have rarely been examined together in the same patients to determine their potentially additive value.To determine whether two previously validated plasma markers (transforming growth factor β1 [TGFβ1] and interleukin-6 soluble receptor [IL6-SR]) and two validated tissue scores (the Genomic Evaluators of Metastatic Prostate Cancer [GEMCaP] and cell cycle progression [CCP] scores) can improve on clinical parameters in predicting adverse pathology after prostatectomy, and how much they vary within tumors with heterogeneous Gleason grade.A case-control study was conducted among men with low-risk cancers defined by biopsy grade group (GG) 1, prostate-specific antigen (PSA) ≤10 ng/mL, and clinical stage ≤ T2 who underwent immediate prostatectomy. We collected paraffin-fixed prostatectomy tissue and presurgical plasma samples from 381 cases from the University of California, San Francisco, and 260 cases from the University of Washington.Pathologic outcomes were minor upgrading/upstaging (GG 2 or pT3a) or major upgrading/upstaging (GG ≥ 3 or ≥ pT3b), and multinomial regression was performed to determine putative markers' ability to predict these outcomes, controlling for PSA, percent of positive biopsy cores, age, and clinical site. For upgraded tumors, a secondary analysis of the GEMCaP and CCP scores from the higher-grade tumor was also performed to evaluate for heterogeneity.Overall, 357 men had no upgrading/upstaging event at prostatectomy, 236 had a minor event, and 67 had a major event. Neither TGFβ1 nor IL6-SR was statistically significantly associated with any upgrading/upstaging. On the contrary, both the CCP and the GEMCaP score obtained from Gleason pattern 3 tissue were directly associated with minor and major upgrading/upstaging on univariate analysis. The two scores correlated with each other, but weakly. On multinomial analysis including both scores in the model, the CCP score predicted minor upgrading/upstaging (odds ratio [OR] 1.62, 95% confidence interval [CI] 1.05-2.49) and major upgrading/upstaging (OR 2.26, 95% CI 1.05-4.90), p = 0.04), and the GEMCaP score also predicted minor upgrading/upstaging (OR 1.05, 95% CI 1.03-1.08) and major upgrading/upstaging (OR 1.07, 95% CI 1.04-1.11), p 0.01). The other clinical parameters were not significant in this model. Among upgraded tumors including both Gleason patterns 3 and 4, both the GEMCaP and the CCP score tended to be higher from the higher-grade tumor. The main limitation was the use of virtual biopsies from prostatectomy tissue as surrogates for prostate biopsies.Biomarker signatures based on analyses of both DNA and RNA significantly and independently predict adverse pathology among men with clinically low-risk prostate cancer undergoing prostatectomy.Validated biomarker scores derived from both prostate cancer DNA and prostate cancer RNA can add independent information to help predict outcomes after prostatectomy.

Prostate cancer is the most commonly diagnosed neoplasm in American men. Although existing biomarkers may detect localized prostate cancer, additional strategies are necessary for improving detection and identifying aggressive disease that may require further intervention. One promising, minimally invasive biomarker is cell-free DNA (cfDNA), which consist of short DNA fragments released into circulation by dying or lysed cells that may reflect underlying cancer. Here we investigated whether differences in cfDNA concentration and cfDNA fragment size could improve the sensitivity for detecting more advanced and aggressive prostate cancer. This study included 268 individuals: 34 healthy controls, 112 men with localized prostate cancer who underwent radical prostatectomy (RP), and 122 men with metastatic castration-resistant prostate cancer (mCRPC). Plasma cfDNA concentration and fragment size were quantified with the Qubit 3.0 and the 2100 Bioanalyzer. The potential relationship between cfDNA concentration or fragment size and localized or mCRPC prostate cancer was evaluated with descriptive statistics, logistic regression, and area under the curve analysis with cross-validation. Plasma cfDNA concentrations were elevated in mCRPC patients in comparison to localized disease (OR5ng/mL = 1.34, P = 0.027) or to being a control (OR5ng/mL = 1.69, P = 0.034). Decreased average fragment size was associated with an increased risk of localized disease compared to controls (OR5bp = 0.77, P = 0.0008). This study suggests that while cfDNA concentration can identify mCRPC patients, it is unable to distinguish between healthy individuals and patients with localized prostate cancer. In addition to PSA, average cfDNA fragment size may be an alternative that can differentiate between healthy individuals and those with localized disease, but the low sensitivity and specificity results in an imperfect diagnostic marker. While quantification of cfDNA may provide a quick, cost-effective approach to help guide treatment decisions in advanced disease, its use is limited in the setting of localized prostate cancer.

PURPOSE Cell-free DNA (cfDNA) may allow for minimally invasive identification of biologically relevant genomic alterations and genetically distinct tumor subclones. Although existing biomarkers may detect localized prostate cancer, additional strategies interrogating genomic heterogeneity are necessary for identifying and monitoring aggressive disease. In this study, we aimed to evaluate whether circulating tumor DNA can detect genomic alterations present in multiple regions of localized prostate tumor tissue. METHODS Low-pass whole-genome and targeted sequencing with a machine-learning guided 2.5-Mb targeted panel were used to identify single nucleotide variants, small insertions and deletions (indels), and copy-number alterations in cfDNA. The majority of this study focuses on the subset of 21 patients with localized disease, although 45 total individuals were evaluated, including 15 healthy controls and nine men with metastatic castration-resistant prostate cancer. Plasma cfDNA was barcoded with duplex unique molecular identifiers. For localized cases, matched tumor tissue was collected from multiple regions (one to nine samples per patient) for comparison. RESULTS Somatic tumor variants present in heterogeneous tumor foci from patients with localized disease were detected in cfDNA, and cfDNA mutational burden was found to track with disease severity. Somatic tissue alterations were identified in cfDNA, including nonsynonymous variants in FOXA1, PTEN, MED12, and ATM. Detection of these overlapping variants was associated with seminal vesicle invasion ( P = .019) and with the number of variants initially found in the matched tumor tissue samples ( P = .0005). CONCLUSION Our findings demonstrate the potential of targeted cfDNA sequencing to detect somatic tissue alterations in heterogeneous, localized prostate cancer, especially in a setting where matched tumor tissue may be unavailable (ie, active surveillance or treatment monitoring).

BackgroundCell-free DNA’s (cfDNA) use as a biomarker in cancer is challenging due to genetic heterogeneity of malignancies and rarity of tumor-derived molecules. Here we describe and demonstrate a novel machine-learning guided panel design strategy for improving the detection of tumor variants in cfDNA. Using this approach, we first generated a model to classify and score candidate variants for inclusion on a prostate cancer targeted sequencing panel. We then used this panel to screen tumor variants from prostate cancer patients with localized disease in bothin silicoand hybrid capture settings.MethodsWhole Genome Sequence (WGS) data from 550 prostate tumors was analyzed to build a targeted sequencing panel of single point and small (in silicoagainst prostate tumor sequences from 5 patients to assess performance against commonly used alternative panel designs. The panel’s ability to detect tumor-derived cfDNA variants was then assessed using prospectively collected cfDNA and tumor foci from a test set 18 prostate cancer patients with localized disease undergoing radical proctectomy.ResultsThe panel generated from this approach identified as top candidates mutations in known driver genes (e.g. HRAS) and prostate cancer related transcription factor binding sites (e.g. MYC, AR). It outperformed two commonly used designs in detecting somatic mutations found in the cfDNA of 5 prostate cancer patients when analyzed in anin silicosetting. Additionally, hybrid capture and 2,500X sequencing of cfDNA molecules using the panel resulted in detection of tumor variants in all 18 patients of a test set, where 15 of the 18 patients had detected variants found in multiple foci.ConclusionMachine learning-prioritized targeted sequencing panels may prove useful for broad and sensitive variant detection in the cfDNA of heterogeneous diseases. This strategy has implications for disease detection and monitoring when applied to the cfDNA isolated from prostate cancer patients.

PurposeProstate cancer is the most commonly diagnosed neoplasm in American men. Although existing biomarkers may detect localized prostate cancer, additional strategies are necessary for improving detection and identifying aggressive disease that may require further intervention. One promising, minimally invasive biomarker is cell-free DNA (cfDNA), which consist of short DNA fragments released into circulation by dying or lysed cells that may reflect underlying cancer. Here we investigated whether differences in cfDNA concentration and cfDNA fragment size could improve the sensitivity for detecting more advanced and aggressive prostate cancer.Materials and MethodsThis study included 268 individuals: 34 healthy controls, 112 men with localized prostate cancer who underwent radical prostatectomy (RP), and 122 men with metastatic castration-resistant prostate cancer (mCRPC). Plasma cfDNA concentration and fragment size were quantified with the Qubit 3.0 and the 2100 Bioanalyzer. The potential relationship between cfDNA concentration or fragment size and localized or mCRPC prostate cancer was evaluated with descriptive statistics, logistic regression, and area under the curve analysis with cross-validation.ResultsPlasma cfDNA concentrations were elevated in mCRPC patients in comparison to localized disease (OR5 ng/mL= 1.34, P = 0.027) or to being a control (OR5 ng/mL= 1.69, P = 0.034). Decreased average fragment size was associated with an increased risk of localized disease compared to controls (OR5bp= 0.77, P = 0.0008).ConclusionThis study suggests that cfDNA concentration and average cfDNA fragment size may provide a quick, cost-effective approach to help determine which patients will benefit most from further screening and/or disease monitoring to help improve prostate cancer outcomes.

Early detection of pancreatic ductal adenocarcinoma (PDAC) is key to improving patient outcomes; however, PDAC is usually diagnosed late. Therefore, blood-based minimally invasive biomarker assays for limited volume clinical samples are urgently needed. A novel miRNA profiling platform (Abcam Fireplex-Oncology Panel) was used to investigate the feasibility of developing early detection miRNA biomarkers with 20 μL plasma from a training set (58 stage II PDAC cases and 30 controls) and two validation sets (34 stage II PDAC cases and 25 controls; 44 stage II PDAC cases and 18 controls). miR-34a-5p [AUC = 0.77; 95% confidence interval (CI), 0.66–0.87], miR-130a-3p (AUC = 0.74; 95% CI, 0.63–0.84), and miR-222-3p (AUC = 0.70; 95% CI, 0.58–0.81) were identified as significantly differentially abundant in plasma from stage II PDAC versus controls. Although none of the miRNAs individually outperformed the currently used serologic biomarker for PDAC, carbohydrate antigen 19-9 (CA19-9), combining the miRNAs with CA 19-9 improved AUCs from 0.89 (95% CI, 0.81–0.95) for CA 19-9 alone to 0.92 (95% CI, 0.86–0.97), 0.94 (95% CI, 0.89–0.98), and 0.92 (95% CI, 0.87–0.97), respectively. Gene set enrichment analyses of transcripts correlated with high and low expression of the three miRNAs in The Cancer Genome Atlas PDAC sample set. These miRNA biomarkers, assayed in limited volume plasma together with CA19-9, discriminate stage II PDAC from controls with good sensitivity and specificity. Unbiased profiling of larger cohorts should help develop an informative early detection biomarker assay for diagnostic settings. Prevention Relevance: Development of minimally invasive biomarker assays for detection of premalignant disease and early-stage pancreatic cancer is key to improving patient survival. This study describes a limited volume plasma miRNA biomarker assay that can detect early-stage resectable pancreatic cancer in clinical samples necessary for effective prevention and clinical intervention.

We aimed to validate GEMCaP (Genomic Evaluators of Metastatic Cancer of the Prostate) as a novel copy number signature predictive of prostate cancer recurrence.We randomly selected patients who underwent radical prostatectomy at Cleveland Clinic or University of Rochester from 2000 to 2005. DNA isolated from the cancer region was extracted and subjected to high resolution array comparative genomic hybridization. A high GEMCaP score was defined as 20% or greater of genomic loci showing copy number gain or loss in a given tumor. Cox regression was used to evaluate associations between the GEMCaP score and the risk of biochemical recurrence.We report results in 140 patients. Overall 38% of patients experienced recurrence with a median time to recurrence of 45 months. Based on the CAPRA-S (Cancer of the Prostate Risk Assessment Post-Surgical) score 39% of the patients were at low risk, 42% were at intermediate risk and 19% were at high risk. The GEMCaP score was high (20% or greater) in 31% of the cohort. A high GEMCaP score was associated with a higher risk of biochemical recurrence (HR 2.69, 95% CI 1.51-4.77) and it remained associated after adjusting for CAPRA-S score and age (HR 1.94, 95% CI 1.06-3.56). The C-index of GEMCaP alone was 0.64, which improved when combined with the CAPRA-S score and patient age (C-index = 0.75).A high GEMCaP score was associated with biochemical recurrence in 2 external cohorts. This remained true after adjusting for clinical and pathological factors. The GEMCaP biomarker could be an efficient and effective clinical risk assessment tool to identify patients with prostate cancer for early adjuvant therapy.

DNA methylation is a known regulator of mRNA expression. Recent studies in cancer have shown that significant proportions of the variability in mRNA expression can be explained by variation in DNA methylation. However, none of these studies have investigated the influence of race on this relationship. Here, we develop poly-CpG site models of gene expression that facilitate the quantification of race-specific effects on the regulatory connection between methylation and transcriptional gene expression. Paired Illumina 450k methylation and RNA-Seq gene expression data from The Cancer Genome Atlas (TCGA) was used to develop poly-CpG site elastic net penalized Poisson regression models of gene expression that account for race-by-CpG interactions for each of the eight TCGA tumor types with African American (AA) representation (>40 tumors each). Race-specific effects were summarized as the percentage of CpGs in a gene model with retained interaction terms. The method was applied to methylation data from an AA radical prostatectomy prostate cancer cohort (n=261) to generate predicted gene expression estimates that were used to perform a mechanistic epigenome-wide association study (EWAS) of Gleason grade (≤3+4 and ≥4+3 tumors). High percentages of variability in mRNA expression can be explained by poly-CpG site models, incorporating race specific effects, with tumor type ranges of 62-90% overall, 64-87% for AAs, and 64-92% for European Americans (EAs). Race played a significant role in the relationship between methylation and mRNA expression, with 86-97% of gene models containing at least one CpG site with a race-specific effect. Further, the median percentage of CpGs in a gene model with race-specific effects ranging from 11-18%. For these race-specific effects, the vast majority (>99%) had the same direction of association between the CpG site and gene expression in both AAs and EAs but with differing magnitudes. Application of this approach identified unique methylation alterations (97%) associated with Gleason grade compared with single site and comb-p region-based EWAS. In summary, this poly-CpG site modelling approach revealed a significant impact of race in the regulatory relationship between methylation and gene expression. The high percentage of genes -and CpG sites within those genes- demonstrating race-specific effects was a general phenomenon observed across all eight tumor types investigated. In addition to revealing this previously underappreciated contribution of race, the application of the predicted gene expression output from these poly-CpG site models to perform a gene-based EWAS of Gleason grade demonstrated that it provides a) complementary information to commonly used EWAS methods and b) direct expression-based mechanistic interpretation of findings from an AA methylation-based EWAS of prostate cancer. Citation Format: Ian M. Loveless, Yalei Chen, Sudha Sadasivan, Indrani Datta, Nilesh Gupta, Pamela Paris, Jia Li, Benjamin A. Rybicki, Albert M. Levin. The impact of race on the relationship between poly-CpG DNA methylation and mRNA expression in cancer with application to epigenome-wide association studies [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 2100.

Background Pan-cancer studies of somatic copy number alterations (SCNAs) have demonstrated common SCNA patterns across cancer types, but despite demonstrable differences in aggressiveness of some cancers by race, pan-cancer SCNA variation by race has not been explored. This study investigated a) racial differences in SCNAs in both breast and prostate cancer, b) the degree to which they are shared across cancers, and c) the impact of these shared, race-differentiated SCNAs on cancer survival. Methods Utilizing data from The Cancer Genome Atlas (TCGA), SCNAs were identified using GISTIC 2.0, and in each tumor type, differences in SCNA magnitude between African Americans (AA) and European Americans (EA) were tested using linear regression. Unsupervised hierarchical clustering of the copy number of genes residing in race-differentiated SCNAs shared between tumor types was used to identify SCNA-defined patient groups, and Cox proportional hazards regression was used to test for association between those groups and overall/progression-free survival (PFS). Results We identified SCNAs that differed by race in breast (n = 58 SCNAs; permutation p − 4) and prostate tumors (n = 78 SCNAs; permutation p = 0.006). Six race-differentiated SCNAs common to breast and prostate found at chromosomes 5q11.2-q14.1, 5q15-q21.1, 8q21.11-q21.13, 8q21.3-q24.3, 11q22.3, and 13q12.3-q21.3 had consistent differences by race across both tumor types, and all six were of higher magnitude in AAs, with the chromosome 8q regions being the only amplifications. Higher magnitude copy number differences in AAs were also identified at two of these race-differentiated SCNAs in two additional hormonally-driven tumor types: endometrial (8q21.3-q24.3 and 13q12.3-q21.3) and ovarian (13q12.3-q21.3) cancers. Race differentiated SCNA-defined patient groups were significantly associated with survival differences in both cancer types, and these groups also differentiated within triple negative breast cancers based on PFS. While the frequency of the SCNA-defined patient groups differed by race, their effects on survival did not. Conclusions This study identified race-differentiated SCNAs shared by two related cancers. The association of SCNA-defined patient groups with survival demonstrates the clinical significance of combinations of these race-differentiated genomic aberrations, and the higher frequency of these alterations in AA relative to EA patients may explain racial disparities in risk of aggressive breast and prostate cancer.

Approximately 15% of men with newly diagnosed prostate cancer have high risk features which increase the risk of recurrence and metastasis. Better predictive biomarkers could allow for earlier detection of biochemical recurrence and change surveillance and adjuvant treatment paradigms. Circulating tumor cells are thought to represent the earliest form of metastases. However, their role as biomarkers in men with high risk, localized prostate cancer is not well defined.Two to 5 months after prostatectomy we obtained blood samples from 37 patients with high risk, localized prostate cancer, defined as stage T3a or higher, Gleason score 8 or greater, or prostate specific antigen 20 ng/ml or greater. Circulating tumor cells were enumerated using a commercial platform. Matched tumor and single circulating tumor cell sequencing was performed.Circulating tumor cells were detected in 30 of 37 samples (81.1%) with a median of 2.4 circulating tumor cells per ml (range 0 to 22.9). Patients with detectable circulating tumor cells showed a trend toward shorter recurrence time (p=0.12). All patients with biochemical recurrence had detectable circulating tumor cells. Androgen receptor over expression was detected in 7 of 37 patients (18.9%). Patients with biochemical recurrence had more circulating tumor cell copy number aberrations (p=0.027). Matched tumor tissue and single circulating tumor cell sequencing revealed heterogeneity.We noted a high incidence of circulating tumor cell detection after radical prostatectomy and shorter time to biochemical recurrence in men with a higher circulating tumor cell burden and more circulating tumor cell copy number aberrations. Genomic alterations consistent with established copy number aberrations in prostate cancer were detectable in circulating tumor cells but often discordant with cells analyzed in bulk from primary lesions. With further testing in appropriately powered cohorts early circulating tumor cell detection could be an informative biomarker to assist with adjuvant treatment decisions.

Improvements in technologies to yield purer circulating tumor cells (CTCs) will enable a broader range of clinical applications. We have previously demonstrated the use of a commercially available cell-adhesion matrix (CAM) assay to capture invasive CTCs (iCTCs). To improve the purity of the isolated iCTCs, here we used fluorescence-activated cell sorting (FACS) in combination with the CAM assay (CAM + FACS). Our results showed an increase of median purity from the CAM assay to CAM + FACS for the spiked-in cell lines and patient samples analyzed from three different metastatic cancer types: castration resistant prostate cancer (mCRPC), non-small cell lung cancer (mNSCLC) and pancreatic ductal adenocarcinoma cancer (mPDAC). Copy number profiles for spiked-in mCRPC cell line and mCRPC patient iCTCs were similar to expected mCRPC profiles and a matched biopsy. A somatic epidermal growth factor receptor (EGFR) mutation specific to mNSCLC was observed in the iCTCs recovered from EGFR+ mNSCLC cell lines and patient samples. Next-generation sequencing (NGS) of spiked-in pancreatic cancer cell line and mPDAC patient iCTCs showed mPDAC common mutations. CAM + FACS iCTC enrichment enables multiple downstream genomic characterizations across different tumor types.

Early cancer diagnosis, especially while the disease is localized and before symptoms appear, results in significantly higher survival rates compared to late-stage diagnosis. At the time of diagnosis, it is also common to find multiple foci within the prostate in men with localized disease. Cell-free DNA (cfDNA) are short DNA fragments released into circulation by dying cells, which may reflect underlying disease biology and simultaneously allow for the identification of genetically distinct tumor subclones. The objectives of this study are to determine if cfDNA concentration can be used to distinguish between healthy individuals from patients with localized or metastatic castration-resistant prostate cancer (mCRPC), and if somatic mutations identified in tumor tissue are detectable in cfDNA. This study included samples from 277 individuals: 41 healthy donors, 112 patients with localized prostate cancer who underwent radical prostatectomy (RP) at UCSF, and 124 mCRPC patients. Whole peripheral blood was collected in EDTA tubes or PAXgene Blood ccfDNA tubes. Blood and matched tissue from adjacent normal seminal vesicles and multiple tumor regions (1-9 samples per patient) were collected from patients undergoing RP. Extraction of cfDNA was performed on double spun plasma using the Qiagen QIAamp Circulating Nucleic Acid Kits. After extraction, the concentration and fragment length distribution were measured with a Bioanalyzer 2100 Instrument. Fifty-seven samples (multiple tumor tissue foci and matched blood) from nine patients with localized disease were subjected to whole exome sequencing, and 22 samples from five patients were subjected to whole genome sequencing. All samples from 14 patients underwent targeted sequencing with a 2.5Mb panel generated via machine learning on TCGA prostate cancer sequence data. Somatic variant calling was performed with Broad Institute's Terra platform (GATK4/MuTect2) for tumor tissue and with the Curio platform for cfDNA to build consensus sequences leveraging duplex unique molecular tags. To estimate tumor fraction in cfDNA, ichorCNA was used to profile low pass whole genome sequence data. Total cfDNA concentration was able to distinguish between healthy and metastatic (p = 0.001) participants, adjusted for age; and also between localized and metastatic groups (p < 0.001), adjusted for age and PSA. Among the patients with localized disease, cfDNA concentration was not associated with age, PSA, Gleason score, or Decipher Score (a 22 gene metastasis risk-predicting RNA gene expression signature), suggesting the potential for this marker to be independent of factors that are commonly used to assess disease burden and risk of progression. Targeted sequencing of prostate tumors resulted in an average of 16 mutations with a range of 1 to 128 mutations per tissue region. The cfDNA allele frequencies for mutations identified in tumor tissue ranged from 0.5% to 20%. Four patients with variant detection in cfDNA also experienced biochemical recurrence. While copy number analysis identified clonal and potentially subclonal alterations in WGS tumor tissue, no alterations were found in WGS cfDNA. Further analysis of potential factors influencing variant detection in cfDNA (adverse pathology, starting amount of DNA, coverage) will be performed. Citation Format: Emmalyn Chen, Clinton L. Cario, Lancelote Leong, Karen Lopez, César Márquez, Carissa Chu, Patricia S. Li, Erica Oropeza, Imelda Tenggara, Janet Cowan, Jeffry P. Simko, Daniel K. Wells, Robin Kageyama, June M. Chan, Terence Friedlander, Rahul Aggarwal, Felix Feng, Pamela L. Paris, Peter R. Carroll, John S. Witte. Assessing the utility of cell-free DNA in identifying prostate cancer and characterizing tumor heterogeneity via targeted, whole exome, and whole genome multi-region sequencing [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 719.

African Americans (AAs) tend to have more aggressive diseases and worse outcomes for many cancer types compared to European Americans (EAs). Large structural changes on chromosomes, such as somatic copy number alterations (SCNAs), are commonly present in many cancers and associated with clinical outcomes. While studies focusing on single tumor types have identified racial differences in SCNA frequencies and their possible association with cancer outcomes, pan-cancer race-differentiated SCNAs and their clinical implications have not been fully explored. Focusing on two of the most common hormonally driven cancers, breast cancer in women and prostate cancer in men, and using data from The Cancer Genome Atlas (TCGA), we first identified recurrent SCNAs using GISTIC2 in AAs and EAs separately within each cancer. For each GISTIC2 identified SCNA region, we calculated the magnitude deviation from the expected null value (i.e. copy number (CN) = 2) using the area under the log2(CN) (cnAUC) for each tumor. Modeling cnAUC as the outcome variable, we identified SCNAs that differed between AAs and EAs in breast cancers (n=58 SCNAs; permutation p Citation Format: Yalei Chen, Sudha M Sadasivan, Ruicong She, Indrani Datta, Kanika Taneja, Chitale Dhananjay, Nilesh Gupta, Melissa B Davis, Lisa A Newman, Craig G Rogers, Pamela L Paris, Jia Li, Benjamin A Rybicki, Albert M Levin. Breast and prostate cancers harbor common somatic copy number alterations that consistently differ by race and are associated with survival [abstract]. In: Proceedings of the Twelfth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2019 Sep 20-23; San Francisco, CA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(6 Suppl_2):Abstract nr B066.

Although initially responsive to androgen signaling inhibitors (ASIs), metastatic castration-resistant prostate cancer (mCRPC) inevitably develops and is incurable. In addition to adenocarcinoma (adeno), neuroendocrine prostate cancer (NEPC) emerges to confer ASI resistance. We have previously combined laser capture microdissection and phage antibody display library selection on human cancer specimens and identified novel internalizing antibodies binding to tumor cells residing in their tissue microenvironment. We identified the target antigen for one of these antibodies as CD46, a multifunctional protein that is best known for negatively regulating the innate immune system. CD46 is overexpressed in primary tumor tissue and CRPC (localized and metastatic; adeno and NEPC), but expressed at low levels on normal tissues except for placental trophoblasts and prostate epithelium. Abiraterone- and enzalutamide-treated mCRPC cells upregulate cell surface CD46 expression. Genomic analysis showed that the CD46 gene is gained in 45% abiraterone-resistant mCRPC patients. We conjugated a tubulin inhibitor to our macropinocytosing anti-CD46 antibody and showed that the resulting antibody-drug conjugate (ADC) potently and selectively kills both adeno and NEPC cell lines in vitro (sub-nM EC50) but not normal cells. CD46 ADC regressed and eliminated an mCRPC cell line xenograft in vivo in both subcutaneous and intrafemoral models. Exploratory toxicology studies of the CD46 ADC in non-human primates demonstrated an acceptable safety profile. Thus, CD46 is an excellent target for antibody-based therapy development, which has potential to be applicable to both adenocarcinoma and neuroendocrine types of mCRPC that are resistant to current treatment.

BACKGROUND Carotenoids are a class of nutrients with antioxidant properties that have been purported to protect against cancer. However, the reported associations between carotenoids and prostate cancer have been heterogeneous and lacking data on interactions with nucleotide sequence variations and genomic biomarkers. OBJECTIVE To examine the associations between carotenoid levels and the risk of high-grade prostate cancer, also considering antioxidant-related genes and tumor instability. METHODS We measured plasma levels of carotenoids and genotyped 20 single nucleotide polymorphisms (SNP) in SOD1, SOD2, SOD3, XRCC1, and OGG1 among 559 men with non-metastatic prostate cancer undergoing radical prostatectomy. We performed copy number analysis in a subset of these men (n = 67) to study tumor instability assessed as Fraction of the Genome Altered (FGA). We examined associations between carotenoids, genotypes, tumor instability and risk of high-grade prostate cancer (Gleason grade ≥ 4 + 3) using logistic and linear regression. RESULTS Circulating carotenoid levels were inversely associated with the risk of high-grade prostate cancer; odds ratios (OR) and 95% confidence intervals (CI) comparing highest versus lowest quartiles were: 0.34 (95% CI: 0.18–0.66) for α-carotene, 0.31 (95% CI: 0.15–0.63) for β-carotene, 0.55 (0.28–1.08) for lycopene and 0.37 (0.18–0.75) for total carotenoids. SNPs rs25489 in XRCC1, rs699473 in SOD3 and rs1052133 in OGG1 modified these associations for α-carotene, β-carotene and lycopene, respectively (P ≤ 0.05). The proportion of men with a high degree of FGA increased with Gleason Score (P

PurposeWe explored the diagnostic use of circulating tumor cells in patients with neoadjuvant bladder cancer using enumeration and next generation sequencing.Materials and methodsA total of 20 patients with bladder cancer who were eligible for cisplatin based neoadjuvant chemotherapy were enrolled in an institutional review board approved study. Subjects underwent blood draws at baseline and after 1 cycle of chemotherapy. A total of 11 patients with metastatic bladder cancer and 13 healthy donors were analyzed for comparison. Samples were enriched for circulating tumor cells using the novel IsoFlux™ System microfluidic collection device. Circulating tumor cell counts were analyzed for repeatability and compared with Food and Drug Administration cleared circulating tumor cells. Circulating tumor cells were also analyzed for mutational status using next generation sequencing.ResultsMedian circulating tumor cell counts were 13 at baseline and 5 at followup in the neoadjuvant group, 29 in the metastatic group and 2 in the healthy group. The concordance of circulating tumor cell levels, defined as low-fewer than 10, medium-11 to 30 and high-greater than 30, across replicate tubes was 100% in 15 preparations. In matched samples the IsoFlux test showed 10 or more circulating tumor cells in 4 of 9 samples (44%) while CellSearch® showed 0 of 9 (0%). At cystectomy 4 months after baseline all 3 patients (100%) with medium/high circulating tumor cell levels at baseline and followup had unfavorable pathological stage disease (T1-T4 or N+). Next generation sequencing analysis showed somatic variant detection in 4 of 8 patients using a targeted cancer panel. All 8 cases (100%) had a medium/high circulating tumor cell level with a circulating tumor cell fraction of greater than 5% purity.ConclusionsThis study demonstrates a potential role for circulating tumor cell assays in the management of bladder cancer. The IsoFlux method of circulating tumor cell detection shows increased sensitivity compared with CellSearch. A next generation sequencing assay is presented with sufficient sensitivity to detect genomic alterations in circulating tumor cells.

Noninvasive biomarkers that detect the activity of important oncogenic drivers could significantly improve cancer diagnosis and management of treatment. The goal of this study was to determine whether 68Ga-citrate (which avidly binds to circulating transferrin) can detect MYC-positive prostate cancer tumors, as the transferrin receptor is a direct MYC target gene. PET imaging paired with 68Ga-citrate and molecular analysis of preclinical models, human cell-free DNA (cfDNA), and clinical biopsies were conducted to determine whether 68Ga-citrate can detect MYC-positive prostate cancer. Importantly, 68Ga-citrate detected human prostate cancer models in a MYC-dependent fashion. In patients with castration-resistant prostate cancer, analysis of cfDNA revealed that all patients with 68Ga-citrate avid tumors had a gain of at least one MYC copy number. Moreover, biopsy of two PET avid metastases showed molecular or histologic features characteristic of MYC hyperactivity. These data demonstrate that 68Ga-citrate targets prostate cancer tumors with MYC hyperactivity. A larger prospective study is ongoing to demonstrate the specificity of 68Ga-citrate for tumors with hyperactive MYC. Implications: Noninvasive measurement of MYC activity with quantitative imaging modalities could substantially increase our understanding of the role of MYC signaling in clinical settings for which invasive techniques are challenging to implement or do not characterize the biology of all tumors in a patient. Moreover, measuring MYC activity noninvasively opens the opportunity to study changes in MYC signaling in patients under targeted therapeutic conditions thought to indirectly inhibit MYC. Mol Cancer Res; 15(9); 1221–9. ©2017 AACR.

Uniform amplification of low-input DNA is important for applications across biology, including single-cell genomics, forensic science, and microbial and viral sequencing. However, the requisite biochemical amplification methods are prone to bias, skewing sequence proportions and obscuring signals relating to copy number. Digital droplet multiple displacement amplification enables uniform amplification but requires expert knowledge of microfluidics to generate monodisperse emulsions. In addition, existing microfluidic methods are tedious and labor intensive for preparing many samples. Here, we introduce rapid-emulsification multiple displacement amplification, a method to generate monodisperse droplets with a hand-held syringe and hierarchical droplet splitter. Although conventional microfluidic devices require >10 min to emulsify a sample, our system requires tens of seconds and yields data of equivalent quality. We demonstrate the approach by using it to accurately measure copy number variation (CNV) in single cancer cells.

Aberrant DNA hypomethylation may play an important role in the growth rate of glioblastoma (GBM), but the functional impact on transcription remains poorly understood. We assayed the GBM methylome with MeDIP-seq and MRE-seq, adjusting for copy number differences, in a small set of non-glioma CpG island methylator phenotype (non-G-CIMP) primary tumors. Recurrent hypomethylated loci were enriched within a region of chromosome 5p15 that is specified as a cancer amplicon and also encompasses TERT, encoding telomerase reverse transcriptase, which plays a critical role in tumorigenesis. Overall, 76 gene body promoters were recurrently hypomethylated, including TERT and the oncogenes GLI3 and TP73. Recurring hypomethylation also affected previously unannotated alternative promoters, and luciferase reporter assays for three of four of these promoters confirmed strong promoter activity in GBM cells. Histone H3 lysine 4 trimethylation (H3K4me3) ChIP-seq on tissue from the GBMs uncovered peaks that coincide precisely with tumor-specific decrease of DNA methylation at 200 loci, 133 of which are in gene bodies. Detailed investigation of TP73 and TERT gene body hypomethylation demonstrated increased expression of corresponding alternate transcripts, which in TP73 encodes a truncated p73 protein with oncogenic function and in TERT encodes a putative reverse transcriptase-null protein. Our findings suggest that recurring gene body promoter hypomethylation events, along with histone H3K4 trimethylation, alter the transcriptional landscape of GBM through the activation of a limited number of normally silenced promoters within gene bodies, in at least one case leading to expression of an oncogenic protein.