Jennifer Noteboom

Circulating microRNAs as stable blood-based markers for cancer detection

Improved approaches for the detection of common epithelial malignancies are urgently needed to reduce the worldwide morbidity and mortality caused by cancer. MicroRNAs (miRNAs) are small (≈22 nt) regulatory RNAs that are frequently dysregulated in cancer and have shown promise as tissue-based markers for cancer classification and prognostication. We show here that miRNAs are present in human plasma in a remarkably stable form that is protected from endogenous RNase activity. miRNAs originating from human prostate cancer xenografts enter the circulation, are readily measured in plasma, and can robustly distinguish xenografted mice from controls. This concept extends to cancer in humans, where serum levels of miR-141 (a miRNA expressed in prostate cancer) can distinguish patients with prostate cancer from healthy controls. Our results establish the measurement of tumor-derived miRNAs in serum or plasma as an important approach for the blood-based detection of human cancer.

Early detection of recurrent prostate cancer with an ultrasensitive chemiluminescent prostate-specific antigen assay.

OBJECTIVES Treatment failure after radical prostatectomy is most commonly heralded by an increase in serum prostate-specific antigen (PSA) to detectable levels. We evaluated the clinical utility of an ultrasensitive chemiluminescent PSA assay. METHODS We evaluated the assay in banked sera obtained from 170 men after radical prostatectomy. Controls consisted of 142 females, 29 men who had undergone cystoprostatectomy without evidence of prostate cancer, and 25 men without evidence of recurrent disease at least 5 years after prostatectomy for organ-confined disease. Lead time to diagnosis of recurrence was based on comparisons with the IMx or Tandem E assays using a cutoff of 0.1 ng/mL (100 pg/mL). RESULTS The biologic level of detection of this assay is 8 pg/mL. Serum PSA levels were undetectable in 82.4% of females, 86.2% of the cystoprostatectomy patients, and 96% of the radical prostatectomy controls. After radical prostatectomy, PSA levels were undetectable at last check in 104 of 168 (61.9%) men. In the 24 men with prostate cancer recurrence, the enhanced sensitivity of 8 pg/mL provided a mean lead time based on conservative calculations of 12.7 to 22.5 months over conventional assays. Thirty-four of the 41 men with detectable PSA levels and no evidence of disease recurrence had PSA levels of 30 pg/mL or less. CONCLUSIONS PSA levels are undetectable in most men who do not have recurrence of disease after radical prostatectomy. Low but detectable serum PSA levels less than or equal to 30 pg/mL can be produced by nonmalignant sources of PSA. PSA assays with enhanced sensitivity can detect recurrent prostate cancer with significant lead time over conventional assays.

Urinary Prostate Specific Antigen Levels after Radical Prostatectomy

It was recently demonstrated that urinary prostate specific antigen (PSA) is discordant with serum PSA in many patients after radical prostatectomy. This observation led to the speculation that elevated urinary PSA in the face of undetectable serum PSA may indicate early disease recurrence. We measured urinary PSA levels in 30 patients who had undergone radical prostatectomy for prostate carcinoma and 7 patients who had undergone cystoprostatectomy for bladder cancer. PSA levels of randomly collected urine samples ranged from 0.00 to 22.9 ng./ml. and 0.01 to 8.37 ng./ml., respectively. There was no correlation among urinary and serum PSA levels, pathological stage or type of operation. In 14 patients who had undergone radical prostatectomy and who had measurable levels of urinary PSA voided specimens were divided into initial stream and end stream voided samples. The PSA levels in the end stream voided samples were significantly less than the initial stream sample in 12 of the 14 patients. In men who had undergone radical prostatectomy urethral swab samples were analyzed for PSA. Of 26 patients 24 had detectable levels of urethral swab PSA (range 0.01 to 39.04 ng./ml., median 0.93 ng./ml.). Urethral swab PSA levels did not correlate with serum PSA values or pathological stage of disease. Of 7 patients who had defunctionalized urethras after radical cystoprostatectomy 5 had significantly elevated PSA in the urethral wash or swab samples (range 4.3 to 24.5 ng./ml.). Immunohistochemical analysis of urethrectomy specimens demonstrated positive staining for PSA in 3 of 4 specimens. We conclude that the major source of urinary PSA following total prostatectomy is the urethra itself rather than residual prostate tissue. Measuring serial urinary PSA appears to have limited value in monitoring patients after radical prostatectomy. Whether this urethral PSA can ever contaminate the serum levels of PSA after radical prostatectomy is currently under investigation.

Serum percent free prostate-specific antigen in metastatic prostate cancer.

OBJECTIVES To define the serum prostate-specific antigen (PSA) isoform profile in patients who have prostate cancer but do not have a prostate gland, that is, men who have had a previous radical prostatectomy (RP) and subsequently persistent disease as evidenced by elevated PSA. PSA can be reliably measured in the serum in two major isoforms: PSA complexed to alpha1-antichymotrypsin and uncomplexed free PSA (fPSA). Multiple investigations have illustrated the usefulness of the free/total PSA proportion (percent fPSA) in differentiating prostate cancer from benign prostate disease in patients who still have their prostate gland in situ. METHODS Sera were evaluated from 52 men who underwent RP and postoperatively had increased PSA. fPSA and total PSA (tPSA) concentrations were determined using the Abbott AxSYM PSA assays. Percent fPSA was calculated for all patients. RESULTS Median tPSA was 5.45 ng/mL (range 0.93 to 214.99). Median fPSA was 0.69 ng/mL (range 0.11 to 54.93); the median percent fPSA was 13.3% (range 3.9% to 62.9%). There were 27 (52%) patients with percent fPSA less than 15%, 25 (48%) patients with greater than 15%, and 7 (13%) with greater than 30%. No significant relationship was found between percent fPSA and grade, stage, and severity of disease. Percent fPSA was significantly increased in patients who received hormonal, radiation, or combination treatment versus those who received no treatment (P = 0.02 to 0.0007). CONCLUSIONS Serum percent fPSA in men after RP with persistent prostate cancer encompasses a wide range of values with no clear stratifying factor or factors. These observations and further serial studies in patients with progressive metastatic disease may be important in determining the mechanism(s) for lower percent fPSA in men with newly diagnosed prostate cancer.

BAYER IMMUNO 1 PSA ASSAY : AN AUTOMATED, ULTRASENSITIVE METHOD TO QUANTITATE TOTAL PSA IN SERUM

The Bayer Immuno 1™ PSA Assay measures total PSA in human serum and demonstrates excellent performance with an interassay CV ≤ 3.4% and a biological detection limit of 0.03 μg/L. No significant interference from common hormonal and chemotherapeutic drugs, kallikrein, prostatic acid phosphatase, and trypsin, or elevated levels of total bilirubin, hemoglobin, triglycerides, and IgG was observed. The 95th percentile values for healthy individuals increased with age from 3.0 μg/L for males 50–59 years and 3.3 μg/L for males 60–69 years, to 4.6 μg/L for males ≥ 70 years. Clinical studies with retrospective samples demonstrated correspondence between serial measurements of PSA and clinical outcome for 98% of 159 prostate cancer patients. Clinical sensitivity for patients with clinical evidence of disease, untreated at the time of specimen draw, increased with increasing stage from 77.5–100%. Specificity of 60–70% for BPH and other benign urogenital diseases was consistent with previous findings. Bayer Immuno 1 PSA Assay values for 2131 specimens from healthy subjects and patients with prostate cancer, BPH, and other malignant and nonmalignant diseases correlated well with the Abbott IMx® PSA Assay over the range 0.0–6,238 μg/L (Y = 1.10 × + 0.02). The Bayer Immuno 1 PSA Assay provides automated ultrasensitive, precise, and equimolar measurement of total PSA in human serum. J. Clin. Lab. Anal. 12:65–74, 1998.

The percentage of free prostate-specific antigen does not predict extracapsular disease in patients with clinically localized prostate cancer before radical prostatectomy.

Objective To determine whether the percentage of free/total prostate‐specific antigen (f/tPSA) can predict the pathological features in patients with clinically localized prostate cancer before radical prostatectomy.

Abstract 804: Disseminated tumor cell heterogeneity and dormancy in prostate cancer.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Prostate cancer (PCa) can remain in the bone marrow for a prolonged period of time (>5 years) while the patient shows no evidence of disease before the cancer eventually recurs. Dormant cancer cells can be detected in bone, the principal metastatic site of PCa, and these bone-homing cancer cells are known as disseminated tumor cells (DTC). Little information is available on the heterogeneity and dormancy of DTC in PCa. In this study, we isolated and compared the gene expression profile of individual DTC (n=45) from the bone marrow of 4 PCa patients with no evidence of disease and 5 patients with advanced disease. Using principle component analysis and cluster analysis of the 1000 most variable genes, we determined the heterogeneity of the DTC population within each patient. To identify a dormancy signature from DTC in the bone marrow and primary PCa cells, we carried out two gene expression analyses: DTC in patients with no evidence of disease vs. those with advanced disease, and primary PCa tissues from patients with a short vs. long dormancy period post radical prostatectomy (8-86 months). Genes associated in other cancers with cellular senescence, cell-cycle inhibition, and dormancy were analyzed. Candidate genes from both gene expression arrays were validated at the protein level by a tissue microarray consisting of 64 primary PCa cases that recurred after either a short or long dormancy period post radical prostatectomy (6-121 months). The identification of heterogeneous gene signatures in DTC and novel proteins that promote dormancy will guide the development of possible biomarkers and therapeutic targets to prevent PCa recurrence, possibly by either eliminating DTC or inhibiting their escape from dormancy. Citation Format: Hung-Ming Lam, Lisly Chery, Ilsa Coleman, Bryce Lakely, Sandy Larson, Roger Coleman, Lisha Brown, Kathy Doan, Jennifer Noteboom, Xiaotun Zhang, Lawrence True, Peter Nelson, Bruce Montgomery, Paul Lange, Linda Snyder, Robert Vessella, Colm Morrissey. Disseminated tumor cell heterogeneity and dormancy in prostate cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 804. doi:10.1158/1538-7445.AM2013-804

Abstract 406: Characterizing the molecular features of the neuroendocrine phenotype in castration resistant prostate cancer.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Metastatic castration-resistant prostate cancer (CRPC) has a poor prognosis and remains a significant therapeutic challenge. Understanding the conversion from hormone sensitive to castration resistance may promote the development of more effective therapies. Recent findings suggest that neuroendocrine (NE) differentiation may be associated with the development of CRPC. Our objective was to characterize the NE phenotype in CRPC. Using specimens obtained at radical prostatectomy and rapid autopsy at the University of Washington, 2 sets of tissue microarrays were made from 50 radical prostatectomies and 155 metastatic sites from 50 autopsy patients who died from CRPC (with up to 4 metastatic sites from each patient). NE markers, including Chromogranin A (CHGA), Neuron specific enolase (NSE) and Synaptophysin (SYN), as well as androgen receptor (AR) and prostate specific antigen (PSA) were analyzed by immunohistochemistry (IHC). To characterize the molecular features of the NE phenotype in CRPC, 78 corresponding metastatic sites were also assessed by Agilent gene expression analysis. IHC revealed that only 2 of 50 primary prostate cancers had >10% CHGA positive cells whereas 7 of 50 primary prostate cancers 1-10% of cells expressed CHGA and all 50 were AR positive. By contrast, 29 of 50 CRPC autopsy patients had at least 1 CHGA+ metastasis; 53 of 155 metastatic sites had >10% CHGA positive cells (11 sites were AR negative), and 7 had 1-10% CHGA positive cells. Compared to primary prostate carcinomas, CRPC metastases had an increase in the frequency of CHGA+ expression (4% vs. 58%, p 10% of cells in each of the CRPC metastases. Of 155 CRPC metastases: 28 were positive for SYN and 47 were positive for NSE. Co-expression of CHGA and SYN was observed in 22 sites from 10 patients (10 sites did not express AR) and co-expression of CHGA, SYN and NSE was observed in 12 sites from 6 patients (6 sites did not express AR). PSA, a surrogate of AR activity, was absent in all NE CRPC tumors that did not express AR. All AR negative sites (12/155) expressed at least one NE marker. Gene expression data were generated from 78 laser captured metastases, which were grouped into 4 categories: 21 CHGA+ sites, 6 CHGA+, SYN+ and AR- sites, 5 CHGA+, SYN+ and AR+ sites, and 40 CHGA-, SYN-, NSE- and AR+ sites. This study is the first extensive analysis of the NE features of CRPC. Our data suggest that a) the NE phenotype (as defined by CHGA expression) is significantly more common in CRPC than in hormone sensitive primary disease, b) NE status from different sites in the same patient can be heterogeneous, and c) the NE phenotype is not necessarily associated with the loss of AR. These molecular studies suggest that evolution from hormone sensitive to castration resistant disease involves emergence of NE characteristics over time that may explain the behavior of true “androgen independent” disease. Citation Format: Xiaotun Zhang, Ilsa Coleman, Roger Coleman, Khanhthy Doan, Martine Roudier, Lisly Chery, Jennifer Noteboom, Celestia Higano, Lawrence D. True, Paul H. Lange, Peter S. Nelson, Robert L. Vessella, Colm Morrissey. Characterizing the molecular features of the neuroendocrine phenotype in castration resistant prostate cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 406. doi:10.1158/1538-7445.AM2013-406