Ultrasensitive Detection of Attomolar Protein Concentrations by Dropcast Single Molecule Assays.

Measurements of very low levels of biomolecules, including proteins and nucleic acids, remain a critical challenge in many clinical diagnostic applications due to insufficient sensitivity. While digital measurement methods such as Single Molecule Arrays (Simoa), or digital ELISA, have made significant advances in sensitivity, there are still many potential disease biomarkers that exist in accessible biofluids at levels below the detection limits of these techniques. To overcome this barrier, we have developed a simple strategy for single molecule counting, dropcast single molecule assays (dSimoa), that enables more target molecules to be counted through increased sampling efficiency and with a simpler workflow. In this approach, beads are simply dropcast onto a microscope slide and dried into a monolayer film for digital signal readout. The dSimoa platform achieves attomolar limits of detection, with an up to 25-fold improvement in sensitivity over Simoa, the current state of the art for ultrasensitive protein detection. Furthermore, due to its simple readout process and improved cost-effectiveness compared to existing digital bioassays, dSimoa increases amenability to integration into point-of-care platforms. As an illustration of the potential utility of dSimoa, we demonstrate its ability to measure previously undetectable levels of Brachyury, a tissue biomarker for chordoma, in plasma samples. With its significantly enhanced sensitivity and simplicity, dSimoa can pave the way toward the discovery of new biomarkers for early disease diagnosis and improved health outcomes.

Multidisciplinary approach is associated with improved survival of hepatocellular carcinoma patients.

BACKGROUND: Given the complexity of managing hepatocellular carcinoma (HCC), a multidisciplinary approach (MDT) is recommended to optimize management of HCC patients. However, evidence suggesting that MDT improves patient outcome is limited. METHODS: We performed a retrospective cohort study of all patients newly-diagnosed with HCC between 2005 and 2013 (n = 6,619). The overall survival (OS) rates between the patients who were and were not managed via MDT were compared in the entire cohort (n = 6,619), and in the exactly matched cohort (n = 1,396). RESULTS: In the entire cohort, the 5-year survival rate was significantly higher in the patients who were managed via MDT compared to that of the patients who were not (71.2% vs. 49.4%, P < 0.001), with an adjusted hazard ratio (HR) of 0.47 (95% confidence interval [CI]; 0.41-0.53). In the exactly matched cohort, the 5-year survival rate was higher in patients who were managed via MDT (71.4% vs. 58.7%, P < 0.001; HR [95% CI] = 0.67 [0.56-0.80]). The survival benefit of MDT management was observed in most pre-defined subgroups, and was especially significant in patients with poor liver function (ALBI grade 2 or 3), intermediate or advanced tumor stage (BCLC stage B or C), or high alphafetoprotein levels (≥200 ng/ml). CONCLUSION: MDT management was associated with improved overall survival in HCC patients, indicating that MDT management can be a valuable option to improve outcome of HCC patients. This warrants prospective evaluations.

T cell and monocyte/macrophage activation markers associate with adverse outcome, but give limited prognostic value in anemic patients with heart failure: results from RED-HF.

BACKGROUND: Activated leukocytes may contribute to the development and progression of heart failure (HF). We investigated the predictive value of circulating levels of stable and readily detectable markers reflecting both monocyte/macrophage and T-cell activity, on clinical outcomes in HF patients with reduced ejection fraction (HFrEF). METHODS: The association between baseline plasma levels of soluble CD163 (sCD163), macrophage migration inhibitory factor (MIF), granulysin, soluble interleukin-2 receptor (sIL-2R), and activated leukocyte cell adhesion molecule (ALCAM) and the primary endpoint of death from any cause or first hospitalization for worsening of HF was evaluated using multivariable Cox proportional hazard models in 1541 patients with systolic HF and mild to moderate anemia, enrolled in the Reduction of Events by darbepoetin alfa in Heart Failure (RED-HF) trial. Modifying effects and interaction with darbepoetin alfa treatment were also assessed. RESULTS: All leukocyte markers, except granulysin, were associated with the primary outcome and all-cause death in univariate analysis (all p < 0.01) and remained significantly associated in multivariable analysis adjusting for conventional clinical variables (e.g. age, gender, BMI, NYHA class, creatinine, LVEF, etiology) and CRP. However, after final adjustment for TnT and NT-proBNP no associations were found with outcomes. No interaction with darbepoetin alpha treatment was observed for any marker. CONCLUSIONS: Leukocyte activation markers sCD163, MIF, sIL-2R, and ALCAM were associated with adverse outcome in patients with HFrEF, but add little as prognostic markers on top of established biochemical risk markers. CLINICAL TRIAL REGISTRATION: https://clinicaltrials.gov/ct2/show/NCT00358215 .

Serum CD166: a novel hepatocellular carcinoma tumor marker.

BACKGROUND: We evaluated the diagnostic value of serum-CD166 in patients with hepatocellular carcinoma (HCC). METHODS: Tissue-CD166 was measured using immunohistochemistry. Cell proliferation and migration were evaluated using MTT and Transwell assays, respectively. Serum-CD166 was examined using ELISA and western blotting. RESULTS: CD166 was up-regulated in HCC compared to those in normal liver tissues. Cell proliferation was positively correlated and cell migration was negatively correlated with endogenous CD166 expression in HCC cells. CD166 inhibition using specific shRNA decreased cell proliferation but increased cell migration. Serum CD166 concentrations were much higher in HCC than in colon cancer, hepatitis B, hepatitis C, cirrhosis, gastric cancer, breast cancer, lung cancer and healthy individuals. Serum CD166 also decreased dramatically after curative surgery. A positive correlation was found between serum CD166 and AFP (R=0.7141, p=0.000). Serum CD166 was also positively correlated with γ-GT, bile acid, ALT, AST, and ALP but was negatively correlated with Alb and pre-Alb. The area under the receiver operating characteristic curve for serum-CD166 was 0.9860, which was better than AFP (AUC-ROC, 0.9354) for the differentiation of HCC patients from healthy individuals, with a cut-off of 261 ng/ml (sensitivity: 100.00%, specificity: 89.41%). CONCLUSION: Serum CD166 is a novel diagnostic tumor marker for HCC.

Complementary metal oxide semiconductor compatible silicon nanowires-on-a-chip: fabrication and preclinical validation for the detection of a cancer prognostic protein marker in serum.

An integrated translational biosensing technology based on arrays of silicon nanowire field-effect transistors (SiNW FETs) is described and has been preclinically validated for the ultrasensitive detection of the cancer biomarker ALCAM in serum. High-quality SiNW arrays have been rationally designed toward their implementation as molecular biosensors. The FET sensing platform has been fabricated using a complementary metal oxide semiconductor (CMOS)-compatible process. Reliable and reproducible electrical performance has been demonstrated via electrical characterization using a custom-designed portable readout device. Using this platform, the cancer prognostic marker ALCAM could be detected in serum with a detection limit of 15.5 pg/mL. Importantly, the detection could be completed in less than 30 min and span a wide dynamic detection range (∼10(5)). The SiNW-on-a-chip biosensing technology paves the way to the translational clinical application of FET in the detection of cancer protein markers.

Increased endothelial and macrophage markers are associated with disease severity and mortality in scrub typhus.

OBJECTIVES: Scrub typhus is endemic in the Asia-Pacific region. Mortality is high even with treatment, and further knowledge of the immune response during this infection is needed. This study was aimed at comparing plasma levels of monocyte/macrophage and endothelial related inflammatory markers in patients and controls in South India and to explore a possible correlation to disease severity and clinical outcome. METHODS: Plasma levels of ALCAM, VCAM-1, sCD163, sCD14, YKL-40 and MIF were measured in scrub typhus patients (n = 129), healthy controls (n = 31) and in infectious disease controls (n = 31), both in the acute phase and after recovery, by enzyme immunoassays. RESULTS: Patients had markedly elevated levels of all mediators in the acute phase, differing from both healthy and infectious disease controls. During follow-up levels of ALCAM, VCAM-1, sCD14 and YKL-40 remained elevated compared to levels in healthy controls. High plasma ALCAM, VCAM-1, sCD163, sCD14, and MIF, and in particular YKL-40 were all associated with disease severity and ALCAM, sCD163, MIF and especially YKL-40, were associated with mortality. CONCLUSIONS: Our findings show that scrub typhus is characterized by elevated levels of monocyte/macrophage and endothelial related markers. These inflammatory markers, and in particular YKL-40, may contribute to disease severity and clinical outcome.

[The fetal proteins in prognosis of development of pneumonia in patients with ischemic stroke].

The searching of laboratory predictors of pneumonia in patients with ischemic stroke is an actual issue. The fetal proteins can be such biomarkers. The study was carried out to determine significance of such fetal proteins as alpha-fetoprotein, cancerous embryonic antigen, CA 19-9, CA 125, CA 15-3, CA 72-4, CYFRA 21-1 for prognosis of development of pneumonia in patients with ischemic stroke. The study included sampling of 216 patients in acute period of ischemic stroke. All patients were measured level of fetal proteins in first day from onset of disease using electrochemiluminescence immunoassay. It is demonstrated that CA 72-4 has the most significance for prognosis of development of pneumonia from all analyzed proteins and complications of ischemic stroke. The probability ratio relatively to other fetal proteins added up to 0.460 (CL 95% 0.267-0.791, p=0.011), to other complications--0.629 (CL 95% 0.433-0.913, p=0.015). The threshold value of CA 72-4 for development of pneumonia added up to 0.82 (CL 95% 0.68-0.96, p=0.011) U/ml. Under lower level of CA 72-4 the risk of development of pneumonia increases. Under higher level of CA 72-4 there is statistical probability of absence of developmnent of pneumonia. The threshold value was lower than reference interval which in the study added up to 0.85-1.42 U/ml. The detection of level of CA 72-4 on first day after onset of stroke in patients can be recommended for establishing of group of high risk of development of pneumonia and implementation of therapeutic activities.

Alfa-fetoprotein secreting ovarian sex cord-stromal tumor.

Ovarian sex cord-stromal tumors are relatively infrequent neoplasms that account for approximately 8% of all primary ovarian tumors. They are a heterogeneous group of neoplasms composed of cells derived from gonadal sex cords (granulosa and Sertoli cells), specialized gonadal stroma (theca and Leydig cells), and fibroblasts. They may show androgenic or estrogenic manifestations. We report such a tumor associated with markedly raised serum alpha-fetoprotein (AFP) levels in a young female presenting with a mass and defeminising symptoms. Serum AFP levels returned to normal on removal of tumor.

Activated leukocyte cell adhesion molecule soluble form: a potential biomarker of epithelial ovarian cancer is increased in type II tumors.

Activated leukocyte cell adhesion molecule (ALCAM) is involved in cell-cell interactions in cancer. Shedding of its ectodomain by the metalloprotease ADAM17/TACE generates a soluble form (sALCAM). Here, we show that serum sALCAM levels were significantly higher in epithelial ovarian cancer (EOC) (p < 0.005) than in controls. The performance of sALCAM as classifier, tested by receiver operating characteristic curve, resulted in an area under the curve (AUC) of 0.8067. Serum sALCAM levels showed direct correlation with Carbohydrate Antigen-125 (CA125/MUC16). Moreover, significantly higher levels were found in type II tumors, even in stage I/II, suggesting that elevated sALCAM is an early feature of aggressive EOC. In addition, sALCAM levels were higher in ascites than in sera, suggesting local processing of ALCAM in the peritoneal cavity. In immunodeficient mice, intraperitoneally implanted with a human EOC cell line, human sALCAM progressively increased in serum and was even higher in the ascites. The biochemical characterization of the sALCAM in EOC sera and ascites, showed two predominant forms of approximately 95 and 65 kDa but no EOC-specific isoform. In addition, full-length transmembrane ALCAM but no soluble form was detected in tumor-derived exosomes found in ascites. Finally, in vitro invasion assays showed that inhibition of ADAM17/TACE activity decreased EOC invasive properties, while opposite effects were mediated by a sALCAM-Fc chimera and by an antibody interfering with ALCAM/ALCAM interactions. Altogether these data suggest that sALCAM is a marker of EOC, which correlates with more aggressive type II tumors, and that ADAM17/TACE activity and sALCAM itself mediate enhanced invasiveness.

A bead-based multiplexed immunoassay to evaluate breast cancer biomarkers for early detection in pre-diagnostic serum.

This study investigates whether a set of ten potential breast cancer serum biomarkers and cancer antigens (osteopontin (OPN), haptoglobin, cancer antigen 15-3 (CA15-3), carcinoembryonic antigen (CEA), cancer antigen 125 (CA-125), prolactin, cancer antigen 19-9 (CA19-9), α-fetoprotein (AFP), leptin and migration inhibitory factor (MIF)) can predict early stage breast cancer in samples collected before clinical diagnosis (phase III samples). We performed a nested case-control study within the Prospect-EPIC (European Prospective Investigation into Cancer and nutrition) cohort. We examined to what extent the biomarker panel could discriminate between 68 women diagnosed with breast cancer up to three years after enrollment and 68 matched healthy controls (all 56-64 years at baseline). Using a quantitative bead-based multiplexed assay, we determined protein concentrations in serum samples collected at enrollment. Principal Component Analysis (PCA) and Random Forest (RF) analysis revealed that on the basis of all ten proteins, early cases could not be separated from controls. When we combined serum protein concentrations and subject characteristics related to breast cancer risk in the RF analysis, this did not result in classification accuracy scores that could correctly classify the samples (sensitivity: 50%, specificity: 50%). Our findings indicate that this panel of selected tumor markers cannot be used for diagnosis of early breast cancer.

ALCAM (CD166) expression and serum levels in pancreatic cancer.

BACKGROUND: This study was conducted to evaluate the expression of the activated leukocyte cell adhesion molecule (ALCAM) in pancreatic cancer (PAC) and to determine whether or not the ectodomain shedding of ALCAM (s-ALCAM) could serve as a biomarker in the peripheral blood of PAC patients. MATERIAL AND METHODS: Tissue specimens and blood sera of patients with PAC (n = 264 and n = 116, respectively) and the sera of 115 patients with chronic pancreatitis (CP) were analyzed via ALCAM immunohistochemistry and s-ALCAM ELISA tests. Results were correlated with clinical, histopathological, and patient survival data (Chi-square test, Kaplan-Meier analysis, log-rank test, respectively). RESULTS: ALCAM was expressed in the majority of PAC lesions. Immunohistochemistry and serum ELISA tests revealed no association between ALCAM expression in primary tumors or s-ALCAM and clinical or histopathological data. Neither ALCAM nor s-ALCAM showed a significant impact regarding overall survival (p = 0.261 and p = 0.660, respectively). S-ALCAM serum levels were significantly elevated compared to the sera of CP patients (p<0.001). The sensitivity of s-ALCAM in detecting PAC was 58.6% at a specificity of 73.9% (AUC = 0.69). CONCLUSIONS: ALCAM is expressed in the majority of PAC lesions, but statistical analysis revealed no association with clinical or pathological data. Although significantly elevated in patients with PAC, the sensitivity and specificity of the s-ALCAM serum quantification test was low. Therefore, its potential as a novel diagnostic marker for PAC remains elusive and further investigations are required.

Detection of activated leukocyte cell adhesion molecule in the serum of breast cancer patients and implications for prognosis.

OBJECTIVE: Conflicting results have been reported about activated leukocyte cell adhesion molecule (ALCAM) expression in breast cancer and its prognostic value. Little is known about the role of ALCAM levels in the serum of breast cancer patients. METHODS: We analyzed soluble ALCAM (sALCAM) levels in the serum of 157 primary breast cancer patients and 48 healthy women by ELISA. In addition, we determined ALCAM protein expression by Western blot analysis (n = 120) and mRNA expression by cDNA microarray analysis (n = 115) in the tumor tissue of corresponding patients. RESULTS: sALCAM levels differed between patients and healthy controls (median 24.2 vs. 18.9 ng/ml, p < 0.001). We observed no correlation between serum levels and protein or mRNA expression in corresponding tumors (r < 0.1, p = n.s.). sALCAM levels were not correlated with histological type, grading, tumor stage, or patient age, but elevated sALCAM levels were associated with shorter disease-free survival (HR = 1.97, 95% CI 1.01-3.2, p = 0.043). CONCLUSIONS: Our results indicate that sALCAM can be detected in the serum of patients with primary breast cancer. Elevated serum levels might indicate more aggressive tumor behavior as they might be an independent factor for a worse prognosis in breast cancer patients.

Relevance of activated leukocyte cell adhesion molecule (ALCAM) in tumor tissue and sera of cervical cancer patients.

BACKGROUND: An altered expression of the activated leukocyte cell adhesion molecule (ALCAM) is associated with cancer progression in various cancer types. In some cancers ALCAM has a prognostic value or is predictive for the benefit of therapeutic interventions. To date there are no data on the role of ALCAM in cervical cancer available. METHODS: In this study, ALCAM expression was analysed by immunohistochemistry (IHC) in tissue samples of 233 patients with cervical cancer, among them 178 with complete follow-up information. In addition, soluble (s-)ALCAM was measured in sera of a subset of the included patients (n = 55) by enzyme-linked immunosorbent assay (ELISA). RESULTS: ALCAM overexpression was detected (immunoreactive score (IRS) 2-12) in 58.4% of the cervical cancer samples. The normal ectocervical or endocervical epithelium showed no ALCAM reactivity. In untreated patients, ALCAM overexpression in tumor tissue tended to be associated with shorter cancer-specific survival (CSS) and disease-free survival (DFS). Patients, whose tumor samples showed ALCAM overexpression receiving a cytotoxic therapy like radiotherapy or chemoradiation, however, had a favourable prognosis compared to those patients, whose cancers showed no or minimal ALCAM staining. This effect was particularly apparent in patients receiving chemoradiation where the CSS was significantly longer in patients with ALCAM-positive tumors (p = 0.038; cumulative incidence rates at 96 months 8%, 95% CI 0%-23%, and 26%, CI 3%-43% in ALCAM-positive and ALCAM-negative cases, respectively).Median preoperative s-ALCAM concentration in sera from tumor patients was 27.6 ng/ml (range 17.5-55.1 ng/ml, mean 28.9 ng/ml), serum levels did not correlate with intratumoral ALCAM expression. CONCLUSIONS: The data of our retrospective study suggest that the prognostic value of ALCAM expression in cervical carcinoma might be therapy-dependent, and that ALCAM might function as a predictive marker for the response to chemoradiation. This should be confirmed in further, prospective studies.

[Hepatic actinomycosis ; presentation a case in a diabetic patient]. / Actinomicosis hepática; presentación de un caso en un paciente diabético.

We report the case of an Hispanic female diabetic patient admitted to our hospital complaining of progressive abdominal pain, weight loss, nausea and vomiting. Work-up included an abdominal computed tomography (CT) scan which reported a large liver mass consistent with atypical abscess. Serum alpha-fetoprotein value was normal, so a fine needle aspiration biopsy of the liver was performed and the report was consistent with an actinomycosis-induced abscess. Patient was treated with intravenous and oral amoxicillin with satisfactory clinical response.

Onco-developmental protein concentrations in the sera of patients with ovarian cancer prior to treatment.

Eight onco-developmental proteins have been measured in the serum of women prior to their operation and treatment for ovarian cancer. Alpha-foetoprotein, CEA, hCG and PAPP-A were not often elevated whereas CPAP, SP1, CanAg 50 and CA 125 were. Many of our findings differed from those of previous workers, possibly due to differences in clinical material and analytical technique. We conclude that large profiles of tumour markers are no longer of use in the investigation of ovarian cancer. At the present time we think that CA 125 could prove the most useful marker in the investigation of ovarian cancer.

Foetal steroid binding protein in British and Japanese women.

In order to examine the newly-discovered sex-steroid binding protein, foetal steroid binding protein (FSBP) in different populations, its binding characteristics and its level were studied by two-tier column ligand binding assay and enzyme-linked immunosorbent assay (ELISA) respectively. In 10 Japanese premenopausal women, analysis of 5 alpha-dihydrotestosterone (DHT) binding in the Cibacron Blue 3GA-Sepharose 6B portion of the column showed a rising plateau pattern with a mean maximum binding of 31.1 +/- 7.41%, whereas of 9 similar British women, 8 displayed unsaturable, non-cooperative binding of 11.6 +/- 8.22% (P less than 0.01). After partial purification of FSBP in these samples, the protein exhibited saturable binding kinetics, median binding 25 (interquartiles 23-34) and 19 (13-25) nmol DHT/l in Japanese and British women, respectively (P less than 0.05). By analyzing FSBP by ELISA in 56 Japanese (45 premenopausal) and 59 British (25 premenopausal) women, higher levels were obtained in the whole Japanese group (P = 0.0016) and in the premenopausal Japanese women (P = 0.018) than in their British counterparts. In both nationalities, FSBP levels were higher in premenopausal women, and there was a significant negative correlation of FSBP with age in both populations, particularly in postmenopausal women. FSBP levels did not correlate with weight, parity, sex hormone binding globulin or albumin levels. The influence of FSBP on free steroid levels remains unclear, but some relationship with ovarian function seems a possibility.

A rapid method for the purification and radioimmunoassay of human alpha-fetoprotein.

Human alpha-fetoprotein (AFP) was isolated from cord serum on an immunoadsorbent column obtained by covalently linking rabbit anti AFP to cyanogen bromide activated Sepharose. Bound AFP was eluted with 8 M urea with better than 50% recovery. The purified AFP was iodinated prior to its use in a double antibody radioimmunoassay. The purification and radioimmunoassay employ commercially available materials. A standard inhibition curve was obtained which allowed determination of AFP levels between 50 and 100 ng/ml in human serum. The assay was verified by measureing AFP levels in normal female serum, pregnancy serum, cord serum, hepatoma ascitic fluid and a standardized AFP solution.

Serum alpha-fetoprotein (AFP) in benign and malignant gastrointestinal diseases: evaluation of an immunoenzymatic assay.

A quantitative immunoenzymatic assay has been developed for alpha-fetoprotein which is sensitive and specific. Seventy-eight percent of United States hepatoma patients have detectable serum alpha-fetoprotein elevations over 50 ng/ml, whereas only 2 of 93 other gastrointestinal tumors were positive. Thirteen percent of patients with acute viral hepatitis, 44% of patients with massive hepatic necrosis, and 23% of patients with chronic active hepatitis had measurable serum alpha-fetoprotein concentrations. However, patients with non-viral acute or chronic liver disease were largely alpha-fetoprotein negative and alpha-fetoprotein was undetectable on multiple postoperative samples from 6 patients after hepatic lobectomy in the rapidly regenerating phase. Therefore, alpha-fetoprotein elevations in nonmalignant liver diseases are not due solely to hepatic regeneration but appear to be related to viral injury. The immunoenzymatic assay does not require purified antigen or radioisotope equipment and can detect and quantitate clinically significant alpha-fetoprotein levels greater than 50 ng/ml.