Phase Assays Research Articles

Clinical impact of molecular profiling in the national prospective cohort Solving Unknown Primary Cancer (SUPER) study.

3056 Background: Cancer of unknown primary (CUP) is a common cause of cancer death, with a median survival < 12 months. SUPER is a prospective cohort study designed to create a national information and biobank of patients (pts) with no confirmed primary site following diagnostic work-up. We aimed to determine the impact of tumour molecular profiling on treatment decisions. Methods: 449 pts were recruited (2013-2021) over 3 phases from 12 Australian sites. Clinical information collected over 12 months included: demographics, treatments, investigations and clinico-pathological characteristics. Molecular tests were centrally performed and included comprehensive panel (CCP) sequencing and gene-expression tissue of origin (TOO) assays in Phase 1&2 and CCP and whole genome sequencing (WGS) in Phase 3. The number of genes reported on increased over the 3 phases. Molecular results were discussed in a molecular tumour board. Clinicians completed clinical management questionnaires before and after receiving molecular results. A combined retrospective evaluation of therapeutic actionability was applied to all DNA sequencing data using the TOPOGRAPH database. Results: Median age 63 years [19-86], with 81% ECOG 0-1 and 87% classified as unfavourable CUP subtype. Reporting timelines and rates of successful sequencing improved over time (Table). Clinically actionable genomic abnormalities were detected in 95/331 (29%) pts. Moderate or high match results on TOO assay were consistent with a suspected or later confirmed TOO as reported by clinicians in 81/195 (42%) pts in phase 1 + 2. Germline pathogenic mutations were detected in 34/331 (10%) pts. Molecular tests confirmed clinician’s treatment (started while awaiting results) was consistent with the most likely TOO in 170/331 (51%) pts. Clinicians reported that molecular tests resulted in a change in management in 11/118 pts (9%) in phase 1 and 40/213 (19%) pts in phases 2+3, with 17/51 (33%) pts changed treatment based on potential TOO determination. 11/51 (22%) pts could access treatment via standard pathways given a more specific tumour-type diagnosis and 15/51 (29%) pts were potentially eligible for clinical trials; however, only 7 pts were well enough for referral. Conclusions: The clinical impact of molecular testing in CUP improved over time as testing became more sophisticated and turnaround times improved. Clinicians suspected TOO pre-profiling was consistent with molecular results in half of the pts. Routine access to novel therapies in CUP remains a challenge. [Table: see text]

Bentracimab demonstrates reversal of antiplatelet effects of ticagrelor: impact of hematocrit and generic versions of ticagrelor in vitro

Abstract Recent clinical studies demonstrated bentracimab provides immediate and sustained reversal of ticagrelor's antiplatelet effects in patients undergoing surgical procedures or with bleeding. Ticagrelor reversal was assessed by VerifyNow PRUTest (VN) and other platelet function testing assays in Phase I-III studies. As decreases in hematocrit (hct) may impact the VN result, it remains to be demonstrated whether this assay can detect platelet function restoration by bentracimab in bleeding patients with low hct. Additionally, bentracimab utility against generic ticagrelors is unknown. The purpose of this study was to determine the impact of decreased hct and generic forms of ticagrelor on platelet function and subsequent reversal by bentracimab, as measured by light transmission aggregometry (LTA) and VN. The hct level in blood samples collected from healthy volunteers (n=10) was sequentially lowered to 32, 30, 25, 22.5, 20 or 15% by addition of autologous platelet-rich plasma to whole blood with matching platelet counts. Prepared samples were treated with nothing, vehicle, or ticagrelor (3.0 μg/mL) followed by treatment with bentracimab (1.2 mg/mL). In a separate study, blood collected from healthy volunteers (n=10) was treated with nothing, vehicle, ticagrelor or generic ticagrelors (3.0 and 5.6 μg/mL; mean and peak plasma levels in the Phase 1 trial) prior to addition of bentracimab (0, 0.6, 1.2, 1.8 and 2.4 mg/mL; below the reported minimum to peak plasma level) to demonstrate a dose response. Reversal was assessed by either ADP-mediated LTA or VN or both assays. Results show a decrease in hct to <30% significantly increased the PRU in untreated samples (p<0.05). Ticagrelor treatment significantly decreased the PRU in all hct groups (p<0.0001). When normalized to each donor's baseline PRU values, bentracimab reversed the ticagrelor effects within 10-min, restoring the PRU value to 54–73% for all hct groups. PRU values were further restored to 85–93% of baseline PRU after 1-hr treatment. The PRU inhibition for both 10-min and 1-hr treatments were not significantly different across the hct groups, except for the 10-min treatment in the 15% hct group vs. unadjusted hct group (p<0.05), suggesting equivalent reversal by bentracimab across the hct range. In the second study, treatment with generic ticagrelor demonstrated significant platelet inhibition and was comparable to standard ticagrelor. Bentracimab (>0.6 mg/mL) immediately restored platelet function after ticagrelor treatment to 80–100% of baseline using the VN test. LTA data demonstrated similar results to VN, but as expected, reversal was less robust with 5.6 μg/mL ticagrelor + 0.6 mg/mL bentracimab combination (mean 55% of baseline values). Collectively, these data suggest the VN test can effectively measure the antiplatelet effects of ticagrelor and subsequent reversal, even at low hct, and bentracimab is also capable of reversing the antiplatelet effects of generic forms of ticagrelor. Funding Acknowledgement Type of funding sources: Private company. Main funding source(s): PhaseBio Pharmaceuticals, Inc

JAK selectivity and the implications for clinical inhibition of pharmacodynamic cytokine signalling by filgotinib, upadacitinib, tofacitinib and baricitinib

ObjectiveJanus kinase inhibitors (JAKinibs) are efficacious in rheumatoid arthritis (RA) with variable reported rates of adverse events, potentially related to differential JAK family member selectivity. Filgotinib was compared with baricitinib,...

Rise and fall of high-sensitivity cardiac troponin T after coronary artery bypass grafting surgery in relation to current definitions of myocardial infarction: a systematic review and meta-analysis

Abstract Background Cardiac troponins are the preferred biomarkers for the diagnosis of myocardial infarction (MI) and play an important role in coronary artery bypass grafting (CABG) related MI (type 5 MI). Different cut-off values, as defined by multiples of the 99th percentile upper reference limit (URL) for isolated and non-isolated troponin elevations have been proposed. Also, these definitions are based on arbitrarily chosen and varying values (3rd and 4th Universal definition: 10xURL, Society for Cardiovascular Angiography and Interventions (SCAI): 35 and 70xURL) and electrocardiographical or imaging findings. In addition to the controversy in MI definitions, the introduction of high-sensitivity cardiac troponin (hs-cTn) assays and their subsequent lower thresholds warrant a thorough re-assessment of hs-cTn dynamics after CABG. Purpose To evaluate the rise and fall of hs-cTn after CABG in relation to the definitions of myocardial infarction. Methods Studies published between 2008 and 2020 reporting hs-cTn concentrations in relation to CABG were searched through Pubmed and reviewed by 2 independent screeners. The search terms were “coronary artery bypass” AND “high-sensitivity cardiac troponin”, including alternative names and abbreviations. Inclusion criteria were the use of a hs-cTn assay in the postoperative phase, either for cardiac troponin T or I. This study was performed in agreement with the PRISMA guidelines. Results Out of 37 screened studies, 15 studies were included (2860 patients). The overall preoperative hs-cTnT concentration (median, 25th-75th percentile) was 43.5 (16.3–57.9) ng/L. Subsequently, in >80% of CABG patients, preoperative hs-cTnT was elevated >14 ng/L. Hs-cTnT was highly variable over time, with a peak of 408.7 (14.9–717.2) ng/L 6–8 h after CABG. Postoperative peak hs-cTnT concentrations rose >10xURL (Universal definition) in 100% of CABG patients. Peak hs-cTnT >35xURL and ±20% >70xURL (SCAI definition) during the first 48 h was 30% and ∼20%, respectively (estimated from data depicted in Figure 1). Electrocardiographic and imaging findings were available for only 1 study (554 patients). Overall 30-day mortality was 1.7 (0.8–4.0)%, reported by 7 studies (2291 patients). Data regarding the association of hs-cTnT and clinical outcome was reported by 1 study. As only 4 studies (217 patients) reported on the results of hs-cTnI, using assays from different vendors, a meta-analysis for hs-cTnI could not be performed. Conclusion The different definitions of type 5 MI propose arbitrarily chosen cTn cut-off values, varying enormously between the definitions. The current thresholds may not apply to the majority of CABG patients, since >80% had elevated preoperative hs-cTnT. Furthermore, hs-cTnT obtained within 48 h after CABG is highly variable over time. Therefore, further research is needed to establish clinically relevant hs-cTn thresholds and timing of sampling for a more accurate diagnosis of CABG procedure related MI. Figure 1 Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): ZonMw Veni grant

Zebra Fish in Toxicology Research: Streptavidin Conjugated Peroxidase Assay in the Development Phase of Zebrafish Embryos to Study Liver Toxicities

Zebrafish have similar hepatic anatomy and cellular architecture just like mammals. Therefore, a number of investigators are using zebrafish to study liver pathologies. However, the evaluation model specific to liver toxicities in zebra fish was not clearly stated earlier. The present study was designed to develop a model of embryonic liver toxicity using dexamethasone (DEXA, 0-20 μM) as a standard hepatotoxic agent. such toxicities are easily measured by streptavidin-conjugated peroxidase assay after 48- hour post-fertilization (hpf) of DEXA treatment. In addition to morphological toxicities at different hpf, DEXA showed significant (*p< 0.05 &**p<0.01) reduction of peroxidase-chromogenic dye reaction in the assay as compared to DEXA untreated embryos at 10 & 20 μM concentration that concluded the hepatocellular toxicity of dexamethasone. Hopefully, the developed model for hepatotoxicity evaluation will be a promising model for the evaluation of new drugs or chemicals as an easy vertebrate model before the commencement of another animal model.

Profile of the therascreen® EGFR RGQ PCR kit as a companion diagnostic for gefitinib in non-small cell lung cancer

ABSTRACTIntroduction: Gefitinib is recently approved by the US Food and Drug Administration as a first-line treatment for non-small cell lung cancer (NSCLC) patients harboring EGFR mutations. The therascreen® EGFR RGQ PCR Kit is approved as a companion diagnostic to select patients with EGFR exon 19 deletions and L858R mutation for treatment with gefitinib.Areas covered: This article reviews the methods for detecting EGFR mutations, the technology and indication of the therascreen® kit, and the clinical utility of the assay in phase 3 and phase 4 clinical trials. Studies that compared the performance of the therascreen® kit with other assays and assessed the kit’s application in non-tissue samples are also discussed.Expert commentary: The therascreen® kit is a highly sensitive real-time polymerase chain reaction test that provides standardised testing and automated interpretation of EGFR mutation status in formalin-fixed, paraffin-embedded (FFPE) tissue samples. Although not indicated for these applications, the test has also shown utility in detecting uncommon sensitizing EGFR mutations as well as mutations in non-tissue samples.

Synergistic anti-oral cancer effects of UVC and methanolic extracts of Cryptocarya concinna roots via apoptosis, oxidative stress and DNA damage

Purpose Radiation combined with natural products may improve the radiosensitivity of cancer cells. This study investigated the potential of a combined modality treatment with Ultraviolet C (UVC; wavelength range 200–280 nm) and our previously identified anti-oral cancer agent (methanolic extracts of Cryptocarya concinna roots; MECCrt) in oral cancer cells.Materials and methods The mechanism of the possible synergy of UVC and MECCrt was explored in terms of cell viability, cell cycle, apoptosis, reactive oxygen species (ROS), mitochondrial membrane potential (MitoMP), and DNA damage analyses.Results In cell viability (%) at 24 h treatment, the low doses of UVC (14 J/m2) and MECCrt (10 μg/ml) resulted in slight damage to human oral cancer Ca9-22 cells (83.2 and 80.4) but was less harmful to human oral normal HGF-1 cells (93.4 and 91.8, respectively). The combined treatment of UVC and MECCrt (UVC/MECCrt) had a lower viability (54.5%) than UVC or MECCrt alone in Ca9-22 cells but no showed significant change in HGF-1 cells. In Ca9-22 cells, the expression of flow cytometry-based apoptosis (sub-G1 phase, annexin V, and pancaspase assays) was significantly higher in UVC/MECCrt than in UVC or MECCrt alone (p < 0.0001). Using flow cytometry, intracellular ROS levels of UVC/MECCrt and MECCrt alone were higher than for UVC alone. MitoMP change and H2A histone family member X (γH2AX; H2AFX)-based DNA damage were synergistically inhibited and induced by MECCrt/UVC compared to its single treatment in Ca9-22 cells, respectively.Conclusion UVC plus MECCrt treatment had selective killing and synergistic anti-proliferative effects against oral cancer cells involving apoptosis, oxidative stress, and DNA damage. This combination therapy appears to have a great clinical potential against oral cancer cells.

Eplet/Epitope analysis of Anti-HLA antibodies can be Used to determine positive MFI cut off of complement binding antibodies

Aim Detection of HLA specific antibodies (Ab) plays a critical role in monitoring of transplant recipients (TR). Their concentration, epitope specificity, isotype and ability to bind complement represent the main components of analysis. In this study, we have demonstrated that HLA class 1 epitopes recognized by complement binding (C1q+) Ab differ from those recognized by non-complement binding (C1q−) Ab. Methods Analysis of anti-HLA Ab was performed in 21 TR DTT treated serum using single antigen bead (SAB) Luminex-IgG technology. Complement binding activity of Ab was investigated using Luminex-C1q (final concentration 150.0 μ g/ml) solid phase (One Lambda, Inc) assay. HLAMatchmaker computer algorithm was used for HLA-A/B epitope/eplet analysis. Data analysis was performed using ANOVA statistics. Results Difference between HLA eplet pattern(s) recognized by Ab was detected in 18 sera when HLA specificity (MFI values) of complement binding (C1q+) and non-complement binding (C1q−) Ab were compared. Limited epitope number of Ab reactivity was not observed in 2 sera, and one serum showed uncertain eplet pattern. Representative eplet specific Ab analysis in a single serum is shown in the Table 1. The largest MFI values of C1q negative Ab were used for subsequent statistical analysis. In the representative serum this value was 231. Using this approach we were able to determine the MFI values range (280–500) of positive cut off for Ab demonstrating C1q binding. Conclusions In 86% of sera (18/21) tested HLA epitope/eplet restricted pattern of Ab reactivity was observed. Functional diversity of reactivity patterns of anti-HLA Ab obtained with Luminex-IgG and Luminex-C1q assays has been demonstrated. C1q positive MFI cut off range of 280–500 can be used in order to better identify complement binding Ab in TR serum. Download : Download full-size image

Detection of Post-Transplant Anti-HLA Donor-Specific Antibodies through the Use of Stored Donors’ Cell Lysates and Solid Phase-Based Cross-Matching

Transplant recipients who have sensitizing events such as pregnancies, blood transfusions and previous transplants often develop antibodies directed against human leukocyte antigen (HLA)-molecules of the donors’ organs. These pre-formed donor-specific antibodies (DSA) represent a high risk of organ failure as a consequence of antibody-mediated hyper-acute or acute allograft rejection. In order to select recipients without donor-specific anti- HLA antibodies the complement-dependent cytotoxicity crossmatch assay (CDC-CM) was established as standard procedure more than forty years ago. This assay, however, is characterized by several drawbacks such as a high degree of vitality (at least 90%) required for the target lymphocytes of a given donor. This requirement highly limits its applicability for patients treated with therapeutic antibodies, special drugs or patients who suffer from underlying diseases i.e., especially from type III (immune complex) auto-immune diseases. Furthermore, only DSA which exert complement-fixing activity are detected. As a consequence novel crossmatch procedures which act independently of the complement system and which do not represent functional assays have been generated in the format of flow cytometry (FACS-) or solid phase (ELISA-) assays. Especially solid phase-based assays the outcomes of which are not limited by insufficient cell vitalities have in spite of various environmental disruptive factors been shown to lead to valid results and not to false positive outcomes in contrast to CDC-based cross-matching. Our current results show the superiority of ELISA-based cross-matching in a novel context. Data are provided which show the ELISA-based applicability of long term-stored donors’ materials to demonstrate or exclude the involvement of DSA in a rejection episode by de facto cross-matching and not only by the virtual crossmatch approach i.e., the comparison of the recipients’ anti-HLA antibody specificities with the donors’ historically identified HLA-pheno- and/or genotypes.

Abstract 4819: Evaluation of PTEN status in circulating tumor cells (CTCs) and matched tumor tissue from castrate-resistant prostate cancer (CRPC) patients

Abstract Background: PTEN loss occurs frequently in prostate cancer and may trigger progression to CRPC through activation of the PI3K/AKT pathway. A blood-based assay that determines PTEN status in CRPC patients could enable informed treatment decisions such as the use of a PI3K-targeted therapy. Here we examined the relationship between PTEN status in CTCs and matched archival and fresh tumor biopsies in 43 CRPC patients. Methods: Nucleated cells from CRPC patient blood were plated onto glass slides and subjected to IF staining and CTC identification by high-speed fluorescent scanners at Epic Sciences. CTCs were identified as CK+/CD45- cells with intact DAPI nuclei, and samples with ≥4 CTCs per 2 slides (74%) were then tested for PTEN by FISH. Heterozygous loss was defined as a decrease in PTEN copies (PTEN &amp;lt; CEP10 and &amp;lt; 2 copies) and homozygous loss as zero PTEN copies. PTEN IHC in tissue was stained using CST clone 138G6 and H-scores ≤ 200 counted as loss. Results: Heterozygous or homozygous loss of PTEN by FISH was observed in 16 of 43 patients (37%) by CTC analysis. In addition to loss of PTEN, changes in ploidy were frequently observed and broad heterogeneity seen both within and between patients. The PTEN status in CTCs correlated strongly with the PTEN status in metastatic tissue: All 10 patients that exhibited homozygous PTEN loss in CTCs showed concordant homozygous PTEN loss in fresh biopsies. This correlation extended to three patients that showed correlated mixed homozygous and hemizygous PTEN loss populations in CTCs and tissue. “Drift or change” in PTEN status from archival to fresh biopsies occurred in 15 patients, In 12 of these patients, PTEN status in CTCs was reflective of the status in fresh tissue. Together with PTEN evaluation, the analysis of androgen receptor expression by immunofluorescence and ERG rearrangements by FISH proved feasibility of multiplex biomarker assessment in CTCs. This analysis demonstrated heterogeneity of AR phenotypes and a positive association of ERG rearrangements and PTEN loss in CTCs, consistent with literature reports. Conclusion: Our results illustrate the potential for using CTCs as a non-invasive, real-time biopsy to determine a patient's current PTEN status. PTEN status will be determined using these assays in an ongoing AKT inhibitor Phase II trial. Citation Format: Elizabeth Punnoose, Eric Tucker, Edith Szafer-Glusman, Jin Zhu, Dena Marrinucci, Jessica Louw, Florence Lee, Mikel Kitchen, Natalee Bales, Lukas Amler, Hartmut Koeppen, Premal Patel, Yibing Yan, Ruth Riisnaes, Gerhardt Attard, Johann de Bono. Evaluation of PTEN status in circulating tumor cells (CTCs) and matched tumor tissue from castrate-resistant prostate cancer (CRPC) patients. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4819. doi:10.1158/1538-7445.AM2014-4819

Abstract 2425: Nucleosomal DNA assay development and utility as PoP biomarker.

Abstract Elevated baseline levels of circulating cell-free nucleosomal DNA (nDNA) are observed in some cancer patients and may be associated with clinical outcome and tumor burden. Transient increases in nDNA in response to effective chemotherapy have been observed, indicating nDNA could be a biomarker for apoptosis and could serve as a minimally invasive proof of principal (PoP) biomarker for cancer treatments. The Cell Death Detection ELISA has been used to measure nDNA levels, but the non-quantitative nature of this assay greatly limits its application in clinical studies. Our objectives were to develop a qualified assay for semi-quantitative measurement of nDNA levels in patient plasma samples and utilize the assay in Phase 1 clinical trials to assess its utility. Here we report on nDNA assay development and its application in 2 clinical trials in hematological malignancies. In order to develop a more quantitative assay, we made nDNA standards from healthy-donor blood samples and established their stability using lyophilized plasma. Recovery of freeze-thawed nDNA standard and plasma samples from normal donors and cancer patients were performed. Methods were established for the collection, storage, and analysis of plasma samples. Samples from subjects in Phase 1 dose-escalation studies undergoing treatment with MEDI-551, an anti-CD19 ADCC-enhanced monoclonal antibody (25 subjects, 0.5 - 12 mg/Kg) or moxetumomab pasudotox (CAT-8015), an anti-CD22 immunotoxin (23 subjects, 20 - 60 μg/Kg) were collected at various time points to assess changes in nDNA with treatment. Predose samples were used to assess baseline levels of nDNA for normalizing responses, comparing differences by type of malignancy, and for prognostic purposes. We developed a semi-quantitative, highly reproducible assay with low intra- and inter-plate variability. Although initial sample thaws significantly decreased signal recovery, subsequent thaws did not. Baseline levels of patient nDNA samples were not significantly different across tumor types (DLBCL, FL, CLL, MCL, MM; P=0.1170). The time course of nDNA changes in the first cycle of treatment illustrated potential differences in mechanism of action and dosing for the 2 studies, with short pronounced fold change (FC) increases occurring early in the cycle (Day 2) for MEDI-551 (weekly dosing, cycle 1) compared to prolonged FC increases at Day 5 for CAT-8015 (dosed day 1, 3, 5 every 28 days). A trend of increased nDNA with dose (MEDI-551, P=0.1522; CAT-8015, P=0.2087) and significantly increased FC for end of treatment samples was noted for both studies (MEDI-551, P=0.0438; CAT-8015, P=0.0014). These results indicate that FC increases of nDNA in response to treatment could potentially provide a proof of principle (PoP) biomarker in some clinical oncology studies. In conclusion, we have developed a new semi-quantitative qualified assay for assessing nDNA in blood which has been utilized to support clinical drug development. Citation Format: Xiaoqing (Sarah) Shi, Haifeng Bao, Yuling Wu, Xiaoru Chen, David L. Gold, Theresa M. LaVallee, Patricia A. Burke. Nucleosomal DNA assay development and utility as PoP biomarker. [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 2425. doi:10.1158/1538-7445.AM2013-2425

Evaluation of PTEN status in circulating tumor cells (CTCs) and matched tumor tissue from patients with castrate-resistant prostate cancer (CRPC).

62 Background: PTEN loss occurs frequently in prostate cancer and may trigger progression to CRPC through activation of the PI3K/AKT pathway. A blood-based assay that determines PTEN status in CRPC patients could enable informed treatment decisions such as the use of a PI3K-targeted therapy. We evaluated PTEN status in archived tissue, metastatic bone biopsies, and CTCs from CRPC patients. Here we report the results of PTEN evaluation in CTCs from 42 CRPC patients and matched tissue from 6 patients with more underway. Methods: Nucleated cells from CRPC patient blood were plated onto glass slides and subjected to IF staining and CTC identification by high-speed fluorescent scanners at Epic Sciences. CTCs were identified as CK+/CD45- cells with intact DAPI nuclei, and samples with ≥5 CTCs per 2 slides (74%) were then tested for PTEN by FISH. Partial loss was defined as a decrease in PTEN copies (PTEN &lt; CEP10) and total loss as zero PTEN copies. PTEN IHC in tissue was stained using CST clone 138G6 and H-scores ≤ 200 counted as loss. Results: Partial or total loss of PTEN by FISH was observed in 15 of 31 patients (48%) by CTC analysis. Changes in ploidy were frequently observed and broad heterogeneity seen both within and between patients. Weighted across patients, the most frequently observed abnormal genotypes in CTCs were 2 CEP10/0 PTEN (total loss, diploid, 5.8%), 3 CEP10/0 PTEN (total loss, triploid, 4.5%), and 3 CEP10/3 PTEN (PTEN-normal, triploid, 4.2%). These genotypes were not observed in over 1,000 white blood cells evaluated evenly across 31 patients, demonstrating that cells identified as CTCs on the Epic platform show malignant features not observed in normal cells. PTEN loss was detected by IHC in 6 of 8 (75%) archived patient samples and 7 of 8 (87%) of matched bone biopsies. Where CTCs were available (6 cases), PTEN status was correctly determined as lost in 4 cases, normal in 1 case and borderline in one case. Conclusions: Our results illustrate the high percentage of PTEN loss in CRPC patients and the potential for using CTCs as a non-invasive, real-time biopsy to determine a patient’s PTEN status. PTEN status will be determined using these assays in an upcoming AKT inhibitor Phase II trial.

COMPARISON OF ENZYME IMMUNOASSAY WITH IMMUNOFLUORESCENCE ASSAY FOR THE DIAGNOSIS OF &lt;I&gt;MYCOPLASMA PNEUMONIAE&lt;/I&gt; RESPIRATORY TRACT INFECTIONS IN CHILDREN

Mycoplasma pneumoniae is a frequent cause of acute respiratory infections in both children and adults. Currently, diagnosis of M. pneumoniae infection will based principally on serology and the detection of IgM can provide an early and sensitive diagnosis in children. A variety of commercial immunodiagnostic assays, such as Indirect Enzyme-Linked Immunosorbent (ELISA) assay and Indirect Immunofluorescence (IFA), are now available as serological methods. The Aim of this comparative prospective study, was to compare two different approaches to the rapid detection of Mycoplasma pneumoniae respiratory tract infections among children. For this purpose, a commercial IFA assay for detection of M. pneumoniae-specific IgM antibody in acute-phase sera was used to compare with ELISA assay for specific-IgM antibody and to sought wither the use of either serological assays as a more reliable laboratory diagnosis for Mycoplasma pneumoniae respiratory tract infections in children. This study was designed as a comparative prospective study in which 90 patients (Mean age of the patients in case group was 5.94±2.73 and in control group was 6.51±2.26) of either sexes were included. These patients were classified into two groups: first group (case group), included 45 patients who had been admitted in hospital with diagnosis of respiratory tract infections and the second group (control group), included 45 healthy patients who had no history of respiratory tract infections. Both the groups were age and sex matched. Presence of IgM antibodies to Mycoplasma pneumoniae was assessed by IFA kit assay in both groups and the detection result was compared with result of previous study using ELISA assay. In the case group, 2 (4%) cases out of 45 children were positive for anti-mycoplasma antibody whereas in the control group, all children were negative. All positive case group patients had symptoms of acute pneumonia. 18 (40%) of the patients were diagnosed with bronchial asthma (40%) inclusive of all the two cases diagnosed with Mycoplasma pneumoniae infection. The IgM-positive rate for IFA assay (4%) was lower than in ELISA assay (9%), as compared from both kits. Taking the ELISA assay as a gold standard for the presence of Mycoplasma pneumoniae respiratory tract infections, the sensitivity, specificity and positive and negative predictive values of the IFA assay in comparison to ELISA, were 50 (CI:8.30 to 91.70%), 100 (CI:91.31 to 100%), 100 and 95.34% respectively. The results of IgM IFA assay showed relatively lower positivity than ELISA assay in the early acute phase, the results of IFA undertaken for the detection of M. pneumoniae respiratory tract infections and it is a first study in the Saudia Arabia of its kind from the region reporting such a disease in children using a serological assays as IFA and ELISA assays for comparasion . We therefore conclude that the use of ELISA assay conducted in the country as a more reliable laboratory diagnosis for Mycoplasma pneumoniae respiratory tract infections in children. Further future studies need to be carried out to investigate the use of PCR to allow fast and reliable diagnosis of M. pneumoniae infection during the early phases of infection in children.

Current clinical and laboratory practice for the investigation of the antiphospholipid syndrome: findings from the 2008 Australasian antiphospholipid antibody survey

The antiphospholipid syndrome (APS) is an autoimmune condition characterised by vascular thromboses and/or pregnancy morbidity, and its diagnosis currently requires laboratory evidence for the presence of antiphospholipid antibodies (aPL). aPL are identified using a large number of laboratory procedures based on one of two distinct test processes, namely 'solid' or 'liquid' phase assays. The former include anticardiolipin antibodies (aCL) and anti-beta-2-glycoprotein-I antibodies (aB2GPI), while the latter are centred on clot-based tests used to identify the lupus anticoagulant (LA). Depending on their clinical presentation, affected individuals might be seen by a variety of clinical specialties including general physicians and general practionners, with a potentially wide variation in the aPL assays requested. The current report summarises findings from the '2008 Australasian antiphospholipid antibody survey', a simple 5-step survey that assessed current clinical and laboratory practice in the investigation of APS. The survey was despatched via various clinical and scientific professional bodies. Responses were received from 130 scientific and clinical personnel, primarily haematology based (94/130; 72%) or immunology based (34/130; 26%). Most respondents (97/130; 75%) ordered or recommended tests for solid phase aPL testing, and most also attempted to grade these tests and their isotypes. Most were familiar with aCL and aB2GPI testing, and tended to request primarily IgG and IgM isotypes of these antibodies. Only a small number of respondents requested/recommended IgA isotype testing of these antibodies or the other solid phase aPL assays (e.g., anti-prothrombin). A similar number of respondents (104/130; 80%) also ordered or recommended tests for LA, and most also attempted to grade these tests and their isotypes. Some discipline-related biases were also evident, in that 32/34 (94%) of immunology-based respondents identified that they ordered or recommended specific solid phase tests for aPL, whereas only 62/94 (66%) of haematology-based respondents did so. In contrast, 83/94 (88%) of haematology-based respondents identified that they ordered or recommended specific LA test procedures, whereas only 18/34 (53%) of immunology-based respondents did so. To our knowledge, this report represents the first ever attempt to survey a wide range of clinical and scientific personnel regarding ordering and recommending practices for aPL testing, and provides a snapshot of current clinical and laboratory practice for the investigation of APS in Australia and New Zealand. Most respondents to our survey still consider the immunoglobulin G (IgG) aCL test to be a useful first-line solid phase aPL test, and the dilute Russell's viper venom time (dRVVT) assay to be the most useful LA test.

Sequence Specificity, Conformation, and Recognition by HMG1 Protein of Major DNA Interstrand Cross-links of Antitumor Dinuclear Platinum Complexes

Interactions of high mobility group (HMG) domain proteins with DNA modified by cisplatin plays a role in mechanisms underlying its antitumor activity. A structural motif recognized by HMG domain proteins on cisplatin-modified DNA is a stable, directional bend of the helix axis. In the present work, bending induced in DNA by major adducts of a novel class of antitumor compounds, represented by the formula [¿trans-PtCl(NH(3))(2)¿H(2)N(CH(2))(2-6)NH(2)]Cl(2), was investigated. The oligodeoxyribonucleotide duplexes containing various site-specific interstrand cross-links of these bifunctional dinuclear platinum drugs were purified and characterized by Maxam-Gilbert footprinting, chemical probing, and phasing assay. It was demonstrated that the cross-links of the dinuclear compounds bent the helix much less than those of cisplatin. Gel retardation assay revealed very weak recognition of DNA adducts of dinuclear complexes by HMG1 protein. Hence, the mediation of antitumor properties of dinuclear platinum complexes by HMG domain proteins is unlikely so that polynuclear platinum compounds may represent a novel class of platinum anticancer drugs acting by a different mechanism than cisplatin and its analogues. A further understanding of how polynuclear platinum compounds modify DNA and how these modifications are processed in cells should provide a rational basis for the design of new platinum drugs rather than searching for cisplatin analogues.

Recognising antibacterial hypersensitivity in children.

Adverse reactions to antibacterial agents are not uncommon in children. They are classified as 'immediate' or 'nonimmediate' according to the time interval between drug administration and onset. Immediate reactions occur within 1 hour and are manifested by urticaria and/or angioedema, bronchospasm and anaphylactic shock; immunological reactions are mediated by IgE antibodies. The main nonimmediate reactions (occuring after more than 1 hour) are maculopapular rash, urticaria and serum sickness; T lymphocytes may participate in maculopapular rash. Clinical assessment of such reactions is complex. The patient's history is fundamental; the allergological examination includes in vivo and in vitro tests selected on the basis of the clinical features and the phase of reaction. In the late phase, prick and intradermal tests are sensitive in evaluating beta-lactam allergy. Together with delayed-reading intradermal testing, patch testing seems to be useful in diagnosing maculopapular reactions to systemically administered aminopenicillins. Determination of specific IgE levels is the most common in vitro method for diagnosing immediate reactions. In the acute phase, serum tryptase and urinary N-methylhistamine assays are reliable in diagnosing type I pathogenic mechanisms in immediate reactions. Unfortunately, there are few in vitro tests for evaluating other reactions, and most are not fully validated. In selected cases, provocation tests should be performed.

Chimeric hepatitis B virus core particles as probes for studying peptide-integrin interactions.

An RGD-containing epitope from the foot-and-mouth disease virus (FMDV) VP1 protein was inserted into the e1 loop of the hepatitis B virus core (HBc) protein. This chimeric protein was expressed at high levels in Escherichia coli and spontaneously assembled into virus-like particles which could be readily purified. These fusion particles elicited high levels of both enzyme-linked immunosorbent assay- and FMDV-neutralizing antibodies in guinea pigs. The chimeric particles bound specifically to cultured eukaryotic cells. Mutant particles carrying the tripeptide sequence RGE in place of RGD and the use of a competitive peptide, GRGDS, confirmed the critical involvement of the RGD sequence in this binding. The chimeric particles also bound to purified integrins, and inhibition by chain-specific anti-integrin monoclonal antibodies implicated alpha 5 beta 1 as a candidate cell receptor for both the chimeric particle and FMDV. Some serotypes of FMDV bound to beta 1 integrins in solid- phase assays, and the chimeric particles competed with FMDV for binding to susceptible eukaryotic cells. Thus, HBc particles may provide a simple, general system for exploring the interactions of specific peptide sequences with cellular receptors.

Selective Destruction of Mitochondria by Aziridinylbenzoquinone (AZQ)

Aziridinylbenzoquinone (AZQ or 2, 4-diaziridinyl-3, 6-bis-carboethoxyamino-1, 4-benzoquinone, NSC No. 182986) is a new chemotherapy agent characterized by lipid solubility, high cerebrospinal fluid penetrance and potential as an antiglioma agent.In our laboratory the effects of AZO on 20 human central nervous system glioma-derived tumor cell lines in culture have been studied utilizing both in vitro solid phase and aqueous microcytotoxicity assays. In these in vitro assays a test concentration range over 3 log units (0.1 to 100 μg/ml AZQ) has indicated varying sensitivity of glioma-derived tumor cell lines. Concentrations sufficient to produce greater than 30 percent cytotoxicity ranged from 1.0 yg/ml to 50 yg/ml, being different for each tumor line tested.

The absence of circulating immune complexes in patients with ankylosing spondylitis.

Sera from 50 patients with well-defined ankylosing spondylitis were examined for circulating immune complexes using both a Clq binding (fluid phase) assay and a Raji cell assay. No more than five of the patients assessed had circulating immune complexes by either one of these techniques and none were positive in both. This result is in contrast to the high prevalence in sera from unselected patients with rheumatoid arthritis and systemic lupus used as positive controls.

Background documentation of evaluation of occupational exposure to airborne asbestos.

This review presents background information and literature documentation to supplement the "Recommended Procedures for Sampling and Counting Asbestos Fibers: Procedures for the Evaluation of Occupational Exposure to Airborne Asbestos" prepared by the joint ACGIH-AIHA Aerosol Hazards Evaluation Committee. It reviews the nature of the inhalation hazard associated with asbesots fibers, the sampling and analytic methods which have been used, and a rationale for the selection of the membrane filter sampling-optical phase microscope identification and assay methodology which is recommended.