Eli N. Glezer

Serum complexed and free prostate-specific antigen (PSA) for the diagnosis of the polycystic ovarian syndrome (PCOS)

Abstract Background: Polycystic ovarian syndrome (PCOS) is a common cause of reproductive and metabolic dysfunction. We hypothesized that serum prostate-specific antigen (PSA) may constitute a new biomarker for hyperandrogenism in PCOS. Methods: We conducted a cross-sectional study of 45 women with PCOS and 40 controls. Serum from these women was analyzed for androgenic steroids and for complexed PSA (cPSA) and free PSA (fPSA) with a novel fifth- generation assay with a sensitivity of ~10 fg/mL for cPSA and 140 fg/mL for fPSA. Results: cPSA and fPSA levels were about three times higher in PCOS compared to controls. However, in PCOS, cPSA and fPSA did not differ according to waist-to-hip ratio, Ferriman-Gallwey score, or degree of hyperandrogenemia or oligo-ovulation. In PCOS and control women, serum cPSA and fPSA levels were highly correlated with each other, and with free and total testosterone levels, but not with other hormones. Adjusting for age, body mass index (BMI) and race, cPSA was significantly associated with PCOS, with an odds ratio (OR) of 5.67 (95% confidence interval [CI]: 1.86, 22.0). The OR of PCOS for fPSA was 7.04 (95% CI: 1.65, 40.4). A multivariate model that included age, BMI, race and cPSA yielded an area-under-the-receiver-operating-characteristic curve of 0.89. Conclusions: Serum cPSA and fPSA are novel biomarkers for hyperandrogenism in PCOS and may have value for disease diagnosis.

Abstract 2012: Accurate measurement of free and complexed PSA concentrations in serum of women using a novel technology with fg/mL sensitivity:

Measurement of free and complexed PSA (prostate specific antigen; complexed with ACT) in certain patient groups such as prostatectomy patients and women at risk of breast and ovarian cancer has been limited by the sensitivity of current immunoassay technology (5-30 pg/mL for common clinical analyzers). Fifth generation assays for total PSA with detection limits of 0.05 to 0.1 pg/mL have been described (McDermet 2009; Thaxton 2009; Wilson 2011). We developed a next-generation assay format based on MSD9s MULTI-ARRAY® electrochemiluminescence technology with detection limits of 2 fg/mL (0.002 pg/mL) for complexed PSA (cPSA) and 20 fg/mL for free PSA (fPSA), requiring only 25 μL of serum or plasma per measurement. Assays were calibrated against the WHO standards 96/668 (free PSA) and 98/670 (90% complexed, 10% free PSA). The dynamic range of both assays was three to four orders of magnitude. Typical intra-plate coefficients of variation ranged from 5% to 15%. LLOQ9s of cPSA and fPSA assays were 5 fg/mL and 55 fg/mL, respectively. Spike recovery and dilution linearity were between 80% and 120%. Approximately 100 serum samples from women were evaluated. Free PSA was detectable in 95% of samples and cPSA was detectable in all samples. Multiple literature reports indicate that free PSA might be a marker for early detection of breast cancer; however, the concentrations of many samples were close to or below the detection limits of the assays used for the studies. We tested 48 serum samples from patients with breast cancer and 37 serum samples from apparently healthy women. As the table below shows, there was no clear difference between PSA concentrations in cases and controls. In conclusion, we developed a next-generation assay format that is 100 to 1000 times more sensitive than the currently available clinical PSA assays. This enables accurate determination of free PSA and cPSA concentrations in the serum of females. Citation Format: Galina N. Nikolenko, Martin K. Stengelin, Laukik Sardesai, Eli N. Glezer, Jacob N. Wohlstadter. Accurate measurement of free and complexed PSA concentrations in serum of women using a novel technology with fg/mL sensitivity. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2012. doi:10.1158/1538-7445.AM2015-2012

Effect of age on serum prostate-specific antigen in women

It is now well established that prostate-specific antigen (PSA) is produced by many female tissues, notably the breast [1]. Circulating serum PSA in women is very low (around 1 pg/mL) [2] and cannot be easily measured with third-generation PSA assays. New, fifth-generation PSA assays have made it possible to accurately measure PSA in serum of all women [3]. We used such an assay to quantify complexed PSA (cPSA, the major form of serum PSA, which is bound to alpha 1 antichymotrypsin) in the serum of 69 women, aged 19–45 years, not taking oral contraceptives. Values ranged from 36,000 to 180 fg/mL. After logarithmic transformation, Spearman correlation revealed a negative association of serum PSA with age (Figure 1; Spearman r = −0.310, p = 0.010). From this correlation, we found that serum PSA in women is approximately halved every decade (e.g. at age 20 years, median PSA is around 2300 fg/mL, at age 30 years around 1300 fg/mL, at age 40 years around 700 fg/mL, and at age 50 years around 360 fg/mL). The reason for the apparent decline of serum PSA with age in women is not known, but it is likely related to other hormonal changes with age because the PSA gene is regulated by androgenic and progestational steroids [4]. Alternatively, serum PSA in women may correlate with the size of the breast, the major female organ producing PSA [1, 2]. We also measured serum PSA in 72 serum samples from 50to 70-year-old women with cancer (number of patients in brackets), such as ovarian (22), pancreas (21), lung (20), colorectal (10), and gastrointestinal (9). The cPSA median in these women ranged from 150 to 330 fg/mL, as expected for this age range (Figure 1).

Serum complexed and free prostate specific antigen levels are lower in female elite athletes in comparison to control women

Background: We hypothesize that prostate specific antigen (PSA), a protein that it is under regulation by androgens, may be differentially expressed in female elite athletes in comparison to control women. Methods: We conducted a cross-sectional study of 106 female athletes and 114 sedentary age-matched controls. Serum from these women was analyzed for complexed prostate specific antigen (cPSA) and free prostate specific antigen (fPSA), by fifth generation assays with limits of detection of around 6 and 140 fg/mL, respectively. A panel of estrogens, androgens and progesterone in the same serum was also quantified by tandem mass spectrometry. Results: Both components of serum PSA (cPSA and fPSA) were lower in the elite athletes vs the control group (P=0.033 and 0.013, respectively). Furthermore, estrone (p=0.003) and estradiol (p=0.004) were significantly lower, and dehydroepiandrosterone (p=0.095) and 5-androstene-3β, 17β-diol (p=0.084) tended to be higher in the athletes vs controls. Oral contraceptive use was similar between groups and significantly associated with increased cPSA and fPSA in athletes (p= 0.046 and 0.009, respectively). PSA fractions were not significantly associated with progesterone changes. The Spearman correlation between cPSA and fPSA in both athletes and controls was 0.75 (P < 0.0001) and 0.64 (P < 0.0001), respectively. Conclusions: Elite athletes have lower complexed and free PSA, higher levels of androgen precursors and lower levels of estrogen in their serum than sedentary control women. Abbreviations: cPSA, complexed PSA; fPSA, free PSA; PCOS, polycystic ovarian syndrome; E1, estrone; E2, estradiol; DHEA, dehydroepiandrosterone, Testo, testosterone; DHT, dihydrotestosterone; PROG, progesterone; Delta 4, androstenedione; Delta 5, androst-5-ene-3β, 17β-diol; BMD, body mineral density; LLOQ, lower limit of quantification; ULOQ, upper limit of quantification; LOD, limit of detection; ACT, α 1-antichymotrypsin

Abstract 2241: Serum and plasma IL-17A concentrations in lung cancer patients and controls

Purpose: IL-17A, a cytokine produced by Th17 cells, is reportedly a central regulator of lung tumor growth (Reppert et al., OncoImmunology 1, 2012) and a potential serum biomarker for lung cancer (Xu et al., Biomarkers, 2014). Serum concentrations of IL-17A are too low to measure in most samples (on the order of 0.1 pg/mL) using commercially available ELISAs. In order to overcome this limitation, we developed the S-PLEX™ Human IL-17A ultrasensitive assay which is capable of accurately measuring IL-17A in serum and plasma. We used this assay to measure IL-17A concentrations in serum samples from lung cancer patients and controls. Methods: Serum samples from 59 lung cancer patients and 44 apparently healthy controls (including 14 heavy smokers) were analyzed using S-PLEX technology to determine IL-17A concentrations. In addition, IL-17A concentrations were measured in matched plasma samples from 24 of the controls. S-PLEX is a new ultrasensitive immunoassay format based on MSD9s MULTI-ARRAY® electrochemiluminescence technology. The IL-17A assay requires only 25 μL of serum or plasma per measurement and can be run on the MESO® SECTOR S 600 and MESO QuickPlex® SQ 120 instruments. Results: The S-PLEX Human IL-17A assay was capable of accurately measuring IL-17A concentrations in 125 of the 127 samples tested. The lower limit of detection was 6 fg/mL and the assay range (LLOQ to ULOQ) was from 20 fg/mL to 200,000 fg/mL. Typical intra-plate coefficients of variation (CVs) ranged between 5% and 15%. Control samples run at 3 levels, in multiple replicates over multiple days, produced CVs Conclusion: We have developed a highly specific and sensitive IL-17A assay that is 100 times more sensitive than the current limits of ELISA technology. This assay enables accurate determination of IL-17A concentrations in serum and plasma samples from lung cancer patients and healthy controls. The results from this study do not support the use of IL-17A as an effective serum biomarker for the presence of lung cancer. Citation Format: Animesh Shukla, Sheldon Grove, Simone Barbero, Galina N. Nikolenko, Anu Mathew, Martin K. Stengelin, Eli N. Glezer, Jacob N. Wohlstadter. Serum and plasma IL-17A concentrations in lung cancer patients and controls. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2241.

Abstract 3496: Multiplex panels for cancer-associated biomarkers: quantify 40 biomarkers from 40 μL of serum or plasma.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC There is currently a gap in the translational research between identification of new biomarkers and development and commercialization of FDA-approved diagnostic tests. A large number of potential cancer serum/plasma biomarkers have emerged from academic research projects. Although none of them has sufficient sensitivity and specificity to be used alone as a screening assay, a combination of markers might allow identification of individuals who are at the earliest and most treatable stage of cancer. Discovery and validation of biomarker panels for early detection requires samples, ideally collected before cancer diagnosis, with cases and controls collected using the same protocol. Stand-alone ELISAs require a significant amount of sample, e.g. 100 μL or more per replicate and per assay. To make maximum use of these precious samples, assays must be developed to minimize sample volume requirements. We developed electrochemiluminescence (ECL)-based, multiplexed, serum/plasma immunoassay panels to measure more than 40 cancer-related biomarkers using a 96-well, 7-spot format. Due to the high sensitivity of ECL technology, these assays can be used with diluted serum or plasma bringing the total sample volume required to run all 40 assays down to approximately 40 μL per replicate. These MSD® MULTI-ARRAY assay panels contain most of the classical cancer markers (AFP, Ca125, Ca15.3, Ca19.9, Ca125, CEA, Cyfra 21.1, Her-2, NSE) as well as a number of cancer-associated markers such as growth factors and their receptors (e.g., VEGF, sFlt-1, cMet, SCF, cKit, EGFR); cytokines and chemokines (e.g., IL6, IL-2R); MMPs; and inflammation markers. The assay format is simple: diluted sample or calibrator is added to blocked and washed plates. After a 2-hour incubation with agitation, plates are washed, and detection antibody reagent is added. After a 1-hour incubation, plates are washed and read on an MSD SECTOR® Imager (read time, 1-3 minutes per plate). The assays are sensitive enough to measure these biomarkers in normal samples, and the dynamic range extends beyond the elevated levels expected in disease states. Multiplexing does not affect accuracy; each analyte is measured accurately even when other analytes are present in high abundance. Spike recovery and dilution linearity for most assays were in the range of 80% to 120% of expected values. Detection limits for a majority of the assays were between 1 and 10 pg/mL, and dynamic ranges were between 3 and 4 logs. In conclusion, MSD MULTI-ARRAY® assay panels have been developed and validated for measurement of serum biomarkers of relevance to early detection, diagnosis, prognosis, and/or monitoring of various cancers. The versatility, ease of use, and high-throughput features of the technology make it ideally suited for large scale clinical studies. Citation Format: Martin K. Stengelin, Anahit Aghvanyan, Gelu Dobrescu, Mingyue Wang, Anu Mathew, Eli N. Glezer, Jacob N. Wohlstadter. Multiplex panels for cancer-associated biomarkers: quantify 40 biomarkers from 40 μL of serum or plasma. [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 3496. doi:10.1158/1538-7445.AM2013-3496

Abstract 1163: Detection of hepatocellular carcinoma cases using a multiplex cancer biomarker panel.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related mortality worldwide with a 5-year survival rate of only 14% in the United States. The age-adjusted incidence rate of HCC in the United States is high and rapidly rising (from 1.6 per 100,000 in 1975 to 7.9 in 2009). The only clinically useful serum biomarker for HCC is alpha-fetoprotein (AFP), which has a reported sensitivity of 39 to 64% and specificity of 76 to 91%. This is neither sensitive nor specific enough to be useful, especially since most HCC cases are detected at an advanced state when curative surgery is no longer possible. In this study, the effectiveness of other cancer-related biomarkers in specifically detecting HCC was evaluated using an electrochemiluminescence-based, multiplex serum/plasma immunoassay panel developed on the MSD® platform. An MSD MULTI-ARRAY® 10-plex assay panel (AFP, carcino-embryonic antigen [CEA], cancer antigen 125 [CA125 or Muc-16], carbohydrate antigen 19-9 [CA19-9, sialyl Lewisa], osteopontin [OPN], matrix metalloproteinase 9 [MMP-9], ErbB2, E-cadherin, soluble epidermal growth factor receptor [EGFR], and cKit) was used to screen 25 HCC, 25 cirrhosis (alcohol-induced or due to fatty liver disease), and 30 normal subject serum samples. The assay protocol was simple: a small volume of sample was diluted and added to blocked and washed plates. After a 2-hour incubation with agitation, plates were washed and detection antibody reagent was added. After a 1-hour incubation, plates were washed and read on an MSD SECTOR® Imager 6000 (read time 70 seconds). The levels of AFP, CEA, CA125, and CA19-9 were found to be significantly elevated in the HCC samples compared to levels in the cirrhosis and/or normal samples. The levels of OPN, MMP-9, E-cadherin, and ErbB2 were also significantly altered in the different sample types. Some of these biomarkers, either alone or in combination, were better than AFP alone at distinguishing HCC patients from controls. Combinations of these biomarkers could provide superior performance compared to existing HCC detection modalities. The selected biomarkers must be further evaluated using different sample cohorts to determine effectiveness for detection of HCC cases, particularly in those with different etiologies of development. Early detection of HCC in patients would enable therapeutic intervention at a stage where it would be most effective, significantly reducing mortality rates for HCC, one of the few cancers showing increasing incidence in the United States. Citation Format: Anu Mathew, Eli N. Glezer, Martin Stengelin, Mingyue Wang, Jacob N. Wohlstadter. Detection of hepatocellular carcinoma cases using a multiplex cancer biomarker panel. [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 1163. doi:10.1158/1538-7445.AM2013-1163 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.