Technical comparison of Abbott's UroVysion® and Biocare's CytoFISH urine fluorescence in situ hybridization (FISH) assays.

BACKGROUND: This study aims to compare the technical performance of Abbott's UroVysion and Biocare's CytoFISH urine cytology probe panel and position the CytoFISH probe panel as an alternative to UroVysion. The CytoFISH probe panel was developed based on clinically sensitive chromosomes found to be amplified in bladder cancers, as well as a locus-specific probe also seen to be amplified in bladder tumors. After extensive testing comparing CytoFISH to UroVysion, we present here our findings for the two assays. MATERIALS AND METHODS: A total of 216 cases representing a mix of male (ages 36-99) and female (ages 46-91) patients were assayed with both probe sets. The CytoFISH and UroVysion probe panels were tested in accordance with the UroVysion procedure, as outlined in the manufacturer's supplied package insert with the following exception: the probe volume used was 3µL for UroVysion and 5µL for CytoFISH. RESULTS: The scoring used for the CytoFISH and UroVysion assays revealed a 95% concordance, suggesting that Biocare's CytoFISH Test has at least the same clinical sensitivity and specificity as claimed by the Abbott UroVysion Kit. We found that the CytoFISH 5p15.2 locus-specific probe was easier to score than UroVysion's 9p21 deletion. CONCLUSION: The high rate of concordance between the two assays suggests that Biocare's CytoFISH assay is a robust alternative to Abbott's UroVysion in the diagnosis and monitoring of bladder carcinoma.

How does work environment relate to diagnostic quality? A prospective, mixed methods study in primary care.

OBJECTIVES: The quest to measure and improve diagnosis has proven challenging; new approaches are needed to better understand and measure key elements of the diagnostic process in clinical encounters. The aim of this study was to develop a tool assessing key elements of the diagnostic assessment process and apply it to a series of diagnostic encounters examining clinical notes and encounters' recorded transcripts. Additionally, we aimed to correlate and contextualise these findings with measures of encounter time and physician burnout. DESIGN: We audio-recorded encounters, reviewed their transcripts and associated them with their clinical notes and findings were correlated with concurrent Mini Z Worklife measures and physician burnout. SETTING: Three primary urgent-care settings. PARTICIPANTS: We conducted in-depth evaluations of 28 clinical encounters delivered by seven physicians. RESULTS: Comparing encounter transcripts with clinical notes, in 24 of 28 (86%) there was high note/transcript concordance for the diagnostic elements on our tool. Reliably included elements were red flags (92% of notes/encounters), aetiologies (88%), likelihood/uncertainties (71%) and follow-up contingencies (71%), whereas psychosocial/contextual information (35%) and mentioning common pitfalls (7%) were often missing. In 22% of encounters, follow-up contingencies were in the note, but absent from the recorded encounter. There was a trend for higher burnout scores being associated with physicians less likely to address key diagnosis items, such as psychosocial history/context. CONCLUSIONS: A new tool shows promise as a means of assessing key elements of diagnostic quality in clinical encounters. Work conditions and physician reactions appear to correlate with diagnostic behaviours. Future research should continue to assess relationships between time pressure and diagnostic quality.

Exploring relationships between physician stress, burnout, and diagnostic elements in clinician notes.

OBJECTIVES: To understand the relationship between stressful work environments and patient care by assessing work conditions, burnout, and elements of the diagnostic process. METHODS: Notes and transcripts of audiotaped encounters were assessed for verbal and written documentation related to psychosocial data, differential diagnosis, acknowledgement of uncertainty, and other diagnosis-relevant contextual elements using 5-point Likert scales in seven primary care physicians (PCPs) and 28 patients in urgent care settings. Encounter time spent vs time needed (time pressure) was collected from time stamps and clinician surveys. Study physicians completed surveys on stress, burnout, and work conditions using the Mini-Z survey. RESULTS: Physicians with high stress or burnout were less likely to record psychosocial information in transcripts and notes (psychosocial information noted in 0% of encounters in 4 high stress/burned-out physicians), whereas low stress physicians (n=3) recorded psychosocial information consistently in 67% of encounters. Burned-out physicians discussed a differential diagnosis in only 31% of encounters (low counts concentrated in two physicians) vs. in 73% of non-burned-out doctors' encounters. Burned-out and non-burned-out doctors spent comparable amounts of time with patients (about 25 min). CONCLUSIONS: Key diagnostic elements were seen less often in encounter transcripts and notes in burned-out urgent care physicians.

Anatomy of diagnosis in a clinical encounter: how clinicians discuss uncertainty with patients.

BACKGROUND: Studies consider the clinical encounter as linear, comprising six phases (opening, problem presentation, history-taking, physical examination, diagnosis, treatment and closing). This study utilizes formal conversation analysis to explore patient-physician interactions and understanding diagnostic utterances during these phases. METHODS: This study is a qualitative sub-analysis that explores how the diagnosis process, along with diagnostic uncertainty, are addressed during 28 urgent care visits. We analyzed physicians' hypothesis-generation process by focusing on: location of diagnostic utterances during the encounter; whether certain/uncertain diagnostic utterances were revised throughout the encounter; and how physicians tested their hypothesis-generation and managed uncertainty. We recruited 7 primary care physicians (PCPs) and their 28 patients from Brigham and Women's Hospital (BWH) in 3 urgent care settings. Encounters were audiotaped, transcribed, and coded using NVivo12 qualitative data analysis software. Data were analyzed inductively and deductively, using formal content and conversation analysis. RESULTS: We identified 62 diagnostic communication utterances in 12 different clinical situations. In most (24/28, 86%) encounters, the diagnosis process was initiated before the diagnosis phase (57% during history taking and 64% during physical examination). In 17 encounters (61%), a distinct diagnosis phase was not observed. Findings show that the diagnosis process is nonlinear in two ways. First, nonlinearity was observed when diagnostic utterances occurred throughout the encounter, with the six encounter phases overlapping, integrating elements of one phase with another. Second, nonlinearity was noted with respect to the resolution of diagnostic uncertainty, with physicians acknowledging uncertainty when explaining their diagnostic reasoning, even during brief encounters. CONCLUSIONS: Diagnosis is often more interactive and nonlinear, and expressions of diagnostic assessments can occur at any point during an encounter, allowing more flexible and potentially more patient-centered communication. These findings are relevant for physicians' training programs and helping clinicians improve their communication skills in managing uncertain diagnoses.

Ubiquitin ligase switch in plant photomorphogenesis: A hypothesis.

The E3 ubiquitin ligase COP1 (CONSTITUTIVE PHOTOMORPHOGENIC1) plays a key role in the repression of the plant photomorphogenic development in darkness. In the presence of light, COP1 is inactivated by a mechanism which is not completely understood. This leads to accumulation of COP1's target transcription factors, which initiates photomorphogenesis, resulting in dramatic changes of the seedling's physiology. Here we use a mathematical model to explore the possible mechanism of COP1 modulation upon dark/light transition in Arabidopsis thaliana based upon data for two COP1 target proteins: HY5 and HFR1, which play critical roles in photomorphogenesis. The main reactions in our model are the inactivation of COP1 by a proposed photoreceptor-related inhibitor I and interactions between COP1 and a CUL4 (CULLIN4)-based ligase. For building and verification of the model, we used the available published and our new data on the kinetics of HY5 and HFR1 together with the data on COP1 abundance. HY5 has been shown to accumulate at a slower rate than HFR1. To describe the observed differences in the timecourses of the "slow" target HY5 and the "fast" target HFR1, we hypothesize a switch between the activities of COP1 and CUL4 ligases upon dark/light transition, with COP1 being active mostly in darkness and CUL4 in light. The model predicts a bi-phasic kinetics of COP1 activity upon the exposure of plants to light, with its restoration after the initial decline and the following slow depletion of the total COP1 content. CUL4 activity is predicted to increase in the presence of light. We propose that the ubiquitin ligase switch is important for the complex regulation of multiple transcription factors during plants development. In addition, this provides a new mechanism for sensing the duration of light period, which is important for seasonal changes in plant development.

Degradation of the auxin response factor ARF1.

Auxin-mediated gene expression is largely controlled through a family of DNA-binding proteins known as auxin response factors (ARF). Previous studies on the role of proteolytic regulation in auxin signaling have focused on degradation of their interacting partner, the Aux/IAA proteins. Aux/IAA family members with domain II sequences are rapidly degraded, show auxin-enhanced degradation rates, and interact with the related F-box proteins TIR1 and AFB1-3, which indicates that they are ubiquitylated by a CUL1-dependent E3 ligase. To date, limited data have been generated regarding degradation of ARFs. Here, we focus on the degradation rate of one ARF family member, Arabidopsis thaliana ARF1, and find that the half-lives of N-terminally HA-tagged ARF1 and C-terminally luciferase-tagged ARF1 are both approximately 3-4 h. This half-life appears to be conferred by a component of the middle region (MR), and degradation of the luciferase fusion with the MR is more rapid when the fusion includes an additional nuclear localization signal. ARF1 degradation is proteasome-dependent and rates are not altered in a CUL1 mutant background, suggesting that this ARF is targeted for proteasomal degradation via an alternative set of machinery to that used for Aux/IAA degradation. Consistent with this, exogenous indole acetic acid does not affect the degradation of ARF1. Given increasing evidence that the relative ratio of Aux/IAAs to ARFs rather than the absolute quantity within the cell appears to be the mode through which auxin signaling is modulated, this half-life is likely to be biologically relevant.

Tumor localization and antitumor efficacy of novel sapphyrin compounds.

Sapphyrins are pentapyrrolic metal-free expanded porphyrins with potential medical use as anticancer agents. The novel sapphyrin derivative, PCI-2050, functionalized with 2-[2-(2-methoxyethoxy)ethoxy]ethoxy groups to enhance solubility and a modified bipyrrole moiety was found to be more potent in inducing apoptosis than the previously described sapphyrin PCI-2000. Because some sapphyrins may localize to tumors, we took advantage of the intrinsic fluorescence of these compounds to develop a flow cytometry-based assay to track sapphyrin biodistribution in tumor-bearing mice. Ex vivo analysis of sapphyrin-injected animals revealed that PCI-2050 preferentially localized to tumor, whereas PCI-2000 distributed into normal tissues rather than tumor. PCI-2050 uptake in xenograft tumor cells and resultant tumor cell cytotoxicity was dose dependent. To investigate structure-activity relationships, we focused on PCI-2050 and three derivatives that differ by their alkyl substituents on the bipyrrole moiety: PCI-2051, PCI-2052, and PCI-2053. Treatment of Ramos cells in culture or treatment of Ramos xenograft-bearing animals with each of the sapphyrins followed by ex vivo growth of tumor cells revealed the same pattern of cytotoxicity: PCI-2050 > PCI-2052 > PCI-2051 > PCI-2053. Thus, subtle changes in the alkyl substituents on the bipyrrole moiety result in significant changes in antitumor activity. PCI-2050 displayed significant antitumor efficacy in both Ramos and RKO xenograft models without hematologic, hepatic, or renal abnormalities as assessed by complete blood counts and serum chemistries. On the basis of these findings, it is concluded that the sapphyrin PCI-2050 warrants further evaluation as a potential anticancer agent due to its intrinsic proapoptotic activity and tumor localization ability.

Motexafin gadolinium modulates levels of phosphorylated Akt and synergizes with inhibitors of Akt phosphorylation.

Motexafin gadolinium (MGd, Xcytrin) is a tumor-selective expanded porphyrin that targets oxidative stress-related proteins. MGd treatment of the follicular lymphoma-derived cell line HF-1 resulted in growth suppression and apoptosis whereas MGd treatment of the Burkitt's lymphoma-derived cell line Ramos resulted in growth suppression but not apoptosis. Because phosphorylation status of Akt/protein kinase B is regulated by oxidative stress, we monitored total and phosphorylated Akt (pAkt) in MGd-treated HF-1 and Ramos cells. Levels of pAkt increased within 30 minutes after MGd treatment of HF-1 but after 4 hours began to show a progressive decline to below baseline levels before cells underwent apoptosis. In MGd-treated Ramos cells, pAkt increased approximately 2-fold within 4 hours and remained persistently elevated. Because pAkt activates survival pathways, we determined if MGd-induced cell death could be enhanced by inhibiting phosphorylation of Akt. The addition of specific inhibitors of Akt phosphorylation (Akt inhibitor 1 or SH-5) reduced pAkt levels in MGd-treated HF-1 and Ramos cells and synergistically enhanced MGd-induced cell death. MGd was also evaluated in combination with celecoxib, an inhibitor of Akt phosphorylation, or docetaxel, a microtubule inhibitor that can decrease Akt phosphorylation. The combination of MGd/celecoxib or MGd/docetaxel resulted in decreased Akt phosphorylation and in synergistic cytotoxicity compared with either agent alone. These data point to a potential protective role for pAkt in MGd-induced apoptosis and suggest that MGd activity may be enhanced by combining it with agents that inhibit Akt phosphorylation.

Control of ASPP2/(53BP2L) protein levels by proteasomal degradation modulates p53 apoptotic function.

The p53 pathway is a central mediator of the apoptotic response. ASPP2/(53BP2L) (apoptosis-stimulating protein of p53 2, also known as 53BP2L) enhances apoptosis through selective stimulation of p53 transactivation of proapoptotic target genes. Although the Rb/E2F pathway regulates ASPP2/(53BP2L) transcription, the complex mechanisms controlling ASPP2/(53BP2L) levels and function remain unknown. We now report that proteasomal degradation modulates ASPP2/(53BP2L) protein levels and apoptotic function. Treatment of cells with proteasomal inhibitors, including the clinically utilized proteasomal inhibitor bortezomib, increases ASPP2/(53BP2L) protein but not RNA levels. Likewise, anthracycline-based chemotherapy, which has multiple mechanisms of action, including proteasomal inhibition, increases ASPP2/(53BP2L) protein but not RNA levels. Proteasomal inhibition or anthracycline treatment increases ASPP2/(53BP2L) protein stability and half-life. Furthermore, the central region of the ASPP2/(53BP2L) protein is ubiquitinated as would be expected for a proteasomal substrate. More importantly, small interfering RNA knockdown of ASPP2/(53BP2L) levels attenuated bortezomib-induced apoptosis, and this effect was greater in wild-type p53 cells. Because elevated levels of ASPP2/(53BP2L) are proapoptotic, these results described an important new molecular mechanism that modulates the p53-ASPP2/(53BP2L) apoptotic pathway.

Sapphyrins induce apoptosis in hematopoietic tumor-derived cell lines and show in vivo antitumor activity.

Sapphyrins are pentapyrrolic, metal-free, expanded porphyrins. In the present study, the activity of sapphyrins as anticancer agents in hematopoietic-derived tumor cells was explored. It was found that a dihydroxylated water-soluble sapphyrin derivative (PCI-2000) is a potent inducer of apoptosis in a wide variety of tumor cell lines including lymphoma (Ramos, DHL-4, and HF-1), leukemia (Jurkat and HL-60), and myeloma (8226/S, 1-310, C2E3, and 1-414). PCI-2000 triggers an apoptotic pathway in these tumor cells as shown by release of cytochrome c from mitochondria; activation of caspases 9, 8, and 3; cleavage of the caspase substrate poly(ADP-ribose) polymerase; and Annexin V binding. Apoptosis can be partially inhibited by overexpression of the antiapoptotic protein Bcl-2 or treatment with benzyloxycarbonyl-valine-alanine-aspartic acid-fluoromethylketone, a cell-permeable caspase inhibitor. Both PCI-2000 and PCI-2010, a tetrahydroxy bis-carbamate derivative of PCI-2000, result in increased levels of phosphorylated p38 mitogen-activated protein kinase. Inhibition of p38 mitogen-activated protein kinase phosphorylation resulted in a synergistic increase of PCI-2000 cytotoxicity. PCI-2010 showed less toxicity in mice than PCI-2000 and was active in slowing the growth of Ramos and HL-60 tumor xenografts in nude mice. These results provide preclinical rationale for the further study of sapphyrins for potential use in the treatment of hematopoietic-derived tumors.