Exome Sequencing of Cell-Free DNA from Metastatic Cancer Patients Identifies Clinically Actionable Mutations Distinct from Primary Disease.

The identification of the molecular drivers of cancer by sequencing is the backbone of precision medicine and the basis of personalized therapy; however, biopsies of primary tumors provide only a snapshot of the evolution of the disease and may miss potential therapeutic targets, especially in the metastatic setting. A liquid biopsy, in the form of cell-free DNA (cfDNA) sequencing, has the potential to capture the inter- and intra-tumoral heterogeneity present in metastatic disease, and, through serial blood draws, track the evolution of the tumor genome. In order to determine the clinical utility of cfDNA sequencing we performed whole-exome sequencing on cfDNA and tumor DNA from two patients with metastatic disease; only minor modifications to our sequencing and analysis pipelines were required for sequencing and mutation calling of cfDNA. The first patient had metastatic sarcoma and 47 of 48 mutations present in the primary tumor were also found in the cell-free DNA. The second patient had metastatic breast cancer and sequencing identified an ESR1 mutation in the cfDNA and metastatic site, but not in the primary tumor. This likely explains tumor progression on Anastrozole. Significant heterogeneity between the primary and metastatic tumors, with cfDNA reflecting the metastases, suggested separation from the primary lesion early in tumor evolution. This is best illustrated by an activating PIK3CA mutation (H1047R) which was clonal in the primary tumor, but completely absent from either the metastasis or cfDNA. Here we show that cfDNA sequencing supplies clinically actionable information with minimal risks compared to metastatic biopsies. This study demonstrates the utility of whole-exome sequencing of cell-free DNA from patients with metastatic disease. cfDNA sequencing identified an ESR1 mutation, potentially explaining a patient's resistance to aromatase inhibition, and gave insight into how metastatic lesions differ from the primary tumor.

Effect of computerized prescriber order entry on pharmacy: experience of one health system.

PURPOSE: Pharmacists' satisfaction with a computerized prescriber order-entry (CPOE) system and the impact of CPOE on pharmacy workflows and order verification were investigated. SUMMARY: A mixed-method study was conducted to evaluate the implementation of a CPOE system in three hospitals of a large Michigan-based health system and early user experience with the system. Surveys of pharmacists before (n = 54) and after (n = 42) CPOE implementation indicated that they held generally positive expectations about CPOE prior to and during system implementation and continued to hold positive views about CPOE after several months of system use. In interviews and focus group discussions, pharmacists reported a number of important CPOE benefits, but they also cited challenges related to CPOE provider alerts, uncertainty about medication timing, and the need to support providers by serving as informal CPOE system trainers. Direct observation of pharmacists before and after CPOE implementation indicated decreases in both the rate of order clarification events (from 0.89 to 0.35 per hour, p < 0.001) and the average time spent per hour clarifying orders (from 4.75 to 2.11 minutes, p = 0.008). CONCLUSION: Several months after CPOE implementation, pharmacists indicated that several aspects of their workload had improved, including the process of medication order clarification, their ability to prioritize work, and their ability to move around within the hospital to respond to demand. However, pharmacists also noted that order ambiguity still existed and that the system needed to be optimized to gain efficiencies and increase clarity.

Patterns of care and side effects for patients prescribed methadone for treatment of chronic pain.

OBJECTIVES: This manuscript evaluates physician monitoring practices and incidence of cardiac side effects following initiation of methadone for treatment of chronic pain as compared to patients who began treatment for chronic pain with morphine sustained release (SR). DESIGN: We retrospectively reviewed medical record data on all new initiations of methadone and compared results of physician monitoring practices to patients with new initiations of morphine SR. A standardized chart tool was used to capture clinical data. Data related to health service utilization and clinical diagnoses were obtained from the VA clinical information system. SETTING: A single VA Medical Center in the Pacific Northwest. PATIENTS: Chronic pain patients prescribed methadone (n=92) or morphine (n=90) in the calendar year 2008. RESULTS: There was no difference between patients prescribed methadone versus patients prescribed morphine SR in the likelihood of receiving an electrocardiogram (ECG) prior to initiating medication (53 percent versus 54 percent) or in the year after opioid initiation (37 percent versus 40 percent). The two groups also did not differ in rates of developing prolonged rate-corrected (QTc) intervals (>450 ms) (11 percent versus 17 percent). Seventy-two percent of all patients discontinued their long-acting opioid regimens before 90 days due to adverse effects or insufficient pain relief. CONCLUSION: Despite recommendations for standardized assessment and cardiac risk monitoring, few patients prescribed methadone received an ECG, and this occurred at a rate that did not differ from patients prescribed morphine SR. Patients discontinued both medications at high rates. Further research is needed to evaluate the clinical significance of QTc prolongation in patients treated with methadone.

Androgen and PARP-1 regulation of TRPM2 channels after ischemic injury.

The calcium-permeable transient receptor potential M2 (TRPM2) ion channel was recently demonstrated to have a sexually dimorphic contribution to ischemic brain injury, with inhibition or knockdown of the channel protecting male brain preferentially. We tested the hypothesis that androgen signaling is required for this male-specific cell-death pathway. Additionally, we tested the hypothesis that differential activation of the enzyme poly (ADP-ribose) polymerase-1 (PARP-1) is responsible for male-specific TRPM2 channel activation and neuronal injury. We observed that administration of the TRPM2 inhibitor clotrimazole (CTZ) 2 hours after onset of ischemia reduced infarct volume in male mice and that protection from ischemic damage by CTZ was abolished by removal of testicular androgens (castration; CAST) and rescued by androgen replacement. Male PARP-1 knockout mice had reduced ischemic damage compared with WT mice and inhibition of TRPM2 with CTZ failed to reduce infarct size. Lastly, we observed that ischemia increased PARP activity in the peri-infarct region of male mice to a greater extent than female mice and the difference was abolished in CAST male mice. Data presented in the current study indicate that TRPM2-mediated neuronal death in the male brain requires intact androgen signaling and PARP-1 activity.

Sex differences in the medical care of VA patients with chronic non-cancer pain.

OBJECTIVE: Despite a growing number of women seeking medical care in the veterans affairs (VA) system, little is known about the characteristics of their chronic pain or the pain care they receive. This study sought to determine if sex differences are present in the medical care veterans received for chronic pain. DESIGN: Retrospective cohort study using VA administrative data. SUBJECTS: The subjects were 17,583 veteran patients with moderate to severe chronic non-cancer pain treated in the Pacific Northwest during 2008. METHODS: Multivariate logistic regression assessed for sex differences in primary care utilization, prescription of chronic opioid therapy, visits to emergency departments for a pain-related diagnosis, and physical therapy referral. RESULTS: Compared with male veterans, female veterans were more often diagnosed with two or more pain conditions, and had more of the following pain-related diagnoses: fibromyalgia, low back pain, inflammatory bowel disease, migraine headache, neck or joint pain, and arthritis. After adjustment for demographic characteristics, pain diagnoses, mental health diagnoses, substance use disorders, and medical comorbidity, women had lower odds of being prescribed chronic opioid therapy (adjusted OR [AOR] 0.67, 95% CI 0.58-0.78), greater odds of visiting an emergency department for a pain-related complaint (AOR 1.40, 95% CI 1.18-1.65), and greater odds of receiving physical therapy (AOR 1.19, 95% CI 1.05-1.33). Primary care utilization was not significantly different between sexes. CONCLUSIONS: Sex differences are present in the care female veterans receive for chronic pain. Further research is necessary to understand the etiology of the observed differences and their associations with clinical outcomes.

Patterns and correlates of prescription opioid use in OEF/OIF veterans with chronic noncancer pain.

OBJECTIVES: Little is known about the treatment Operation Enduring Freedom/Operation Iraqi Freedom (OEF/OIF) veterans receive for chronic noncancer pain (CNCP). We sought to describe the prevalence of prescription opioid use, types, and doses of opioids received and to identify correlates of receiving prescription opioids for CNCP among OEF/OIF veterans. DESIGN: Retrospective review of Veterans Affairs (VA) administrative data. SETTING: Ambulatory clinics within a VA regional health care network. PATIENTS: OEF/OIF veterans who had at least three elevated pain screening scores within a 12-month period in 2008. Within this group, those prescribed opioids (N = 485) over the next 12 months were compared with those not prescribed opioids (N = 277). In addition, patients receiving opioids short term (<90 days, N = 284) were compared with patients receiving them long term (≥90 consecutive days, N = 201). RESULTS: Of 762 OEF/OIF veterans with CNCP, 64% were prescribed at least one opioid medication over the 12 months following their index dates. Of those prescribed an opioid, 59% were prescribed opioids short term and 41% were prescribed opioids long term. The average morphine-equivalent opioid dose for short-term users was 23.7 mg (standard deviation [SD] = 20.5) compared with 40.8 mg (SD = 36.1) for long-term users (P < 0.001). Fifty-one percent of long-term opioid users were prescribed short-acting opioids only, and one-third were also prescribed sedative hypnotics. In adjusted analyses, diagnoses of low back pain, migraine headache, posttraumatic stress disorder, and nicotine use disorder were associated with an increased likelihood of receiving an opioid prescription. CONCLUSION: Prescription opioid use is common among OEF/OIF veterans with CNCP and is associated with several pain diagnoses and medical conditions.

Tolerance to the antinociceptive effect of morphine in the absence of short-term presynaptic desensitization in rat periaqueductal gray neurons.

Opioids activate the descending antinociceptive pathway from the ventrolateral periaqueductal gray (vlPAG) by both pre- and postsynaptic inhibition of tonically active GABAergic neurons (i.e., disinhibition). Previous research has shown that short-term desensitization of postsynaptic µ-opioid receptors (MOPrs) in the vlPAG is increased with the development of opioid tolerance. Given that pre- and postsynaptic MOPrs are coupled to different signaling mechanisms, the present study tested the hypothesis that short-term desensitization of presynaptic MOPrs also contributes to opioid tolerance. Twice-daily injections of morphine (5 mg/kg s.c.) for 2 days caused a rightward shift in the morphine dose-response curve on the hot plate test (D(50) = 9.9 mg/kg) compared with saline-pretreated (5.3 mg/kg) male Sprague-Dawley rats. In vitro whole-cell patch-clamp recordings from vlPAG slices revealed that inhibition of evoked inhibitory postsynaptic currents (eIPSCs) by the MOPr-selective agonist [d-Ala(2),N-Me-Phe(4),Gly(5)-ol]-enkephalin was decreased in morphine-tolerant (EC(50) = 708 nM) compared with saline-pretreated rats (EC(50) = 163 nM). However, short-term desensitization of MOPr inhibition of eIPSCs was not observed in either saline- or morphine-pretreated rats. Reducing the number of available MOPrs with the irreversible opioid receptor antagonist, ß-chlornaltrexamine decreased maximal MOPr inhibition with no evidence of desensitization, indicating that the lack of observed desensitization is not caused by receptor reserve. These results demonstrate that tolerance to the antinociceptive effect of morphine is associated with a decrease in presynaptic MOPr sensitivity or coupling to effectors, but this change is independent of short-term MOPr desensitization.

Extracellular signal-regulated kinase 1/2 activation counteracts morphine tolerance in the periaqueductal gray of the rat.

Repeated administration of opioids produces long-lasting changes in micro-opioid receptor (MOR) signaling that underlie behavioral changes such as tolerance. Mitogen-activated protein kinase (MAPK) pathways, including MAPK extracellular signal-regulated kinases (ERK1/2), are modulated by opioids and are known to produce long-lasting changes in cell signaling. Thus, we tested the hypothesis that ERK1/2 activation contributes to the development and/or expression of morphine tolerance mediated by the periaqueductal gray (PAG). Changes in phosphorylated ERK1/2 expression were assessed with confocal microscopy and compared to behavioral measures of tolerance to the antinociceptive effects of chronic morphine administration. Repeated microinjection of morphine into the PAG produced tolerance and caused a significant increase in ERK1/2 phosphorylation, an effect not evident with acute morphine microinjection. Microinjection of the MAPK/ERK kinase inhibitor, 1,4-diamino-2,3-dicyano-1,4-bis(o-aminophenylmercapto)butadiene ethanolate (U0126), into the PAG had no effect on antinociception produced by acute morphine administration. However, repeated coadministration of U0126 and morphine into the PAG blocked ERK1/2 phosphorylation and enhanced the development of morphine tolerance. Coadministration of U0126 with morphine only on the test day also enhanced the expression of morphine tolerance. Administration of the irreversible opioid receptor antagonist beta-chlornaltrexamine blocked the activation of ERK1/2 caused by repeated morphine microinjections, demonstrating that ERK1/2 activation was a MOR-mediated event. In summary, these studies show that chronic morphine administration alters ERK1/2 signaling and that disruption of ERK1/2 signaling enhances both the development and expression of morphine tolerance. Contrary to expectations, these data indicate that ERK1/2 activation opposes the development of morphine tolerance.

Microinjection of the vehicle dimethyl sulfoxide (DMSO) into the periaqueductal gray modulates morphine antinociception.

Dimethyl sulfoxide (DMSO) is commonly used as a solvent for water-insoluble drugs. Given that DMSO has varying cellular and behavioral effects ranging from increased membrane permeability to toxicity, microinjection of DMSO as a vehicle could confound the effects of other drugs. For example, DMSO is often used as a vehicle for studies examining the neurochemical mechanisms underlying morphine antinociception. Given that the ventrolateral periaqueductal gray (vlPAG) plays a major role in morphine antinociception and tolerance, the effects of DMSO on morphine antinociception mediated by the vlPAG needs to be evaluated. The present experiment tested whether co-administration of DMSO (0, 0.2, 2, or 20%) would alter the antinociceptive effect of microinjecting morphine into the vlPAG. DMSO had no effect on nociception when microinjected into the vlPAG alone, but 2% DMSO enhanced morphine potency when co-administered with morphine. In contrast, twice daily microinjections of DMSO (5 or 20%) for two days reduced the potency of subsequent microinjections of morphine into the vlPAG--an effect that persisted for at least one week. A similar rightward shift in the morphine dose-response curve was caused by morphine tolerance. Co-administration of morphine and DMSO during the pretreatment did not cause a greater shift in the morphine dose-response curve compared to morphine pretreated alone. In conclusion, DMSO can alter morphine antinociception following both acute (enhancement) and chronic (inhibition) administration depending on the concentration. These data reinforce the need to be cautious when using DMSO as a vehicle for drug administration.

Tolerance to repeated morphine administration is associated with increased potency of opioid agonists.

Tolerance to the pain-relieving effects of opiates limits their clinical use. Although morphine tolerance is associated with desensitization of mu-opioid receptors, the underlying cellular mechanisms are not understood. One problem with the desensitization hypothesis is that acute morphine does not readily desensitize mu-opioid receptors in many cell types. Given that neurons in the periaqueductal gray (PAG) contribute to morphine antinociception and tolerance, an understanding of desensitization in PAG neurons is particularly relevant. Opioid activity in the PAG can be monitored with activation of G-protein-mediated inwardly rectifying potassium (GIRK) currents. The present data show that opioids have a biphasic effect on GIRK currents in morphine tolerant rats. Opioid activation of GIRK currents is initially potentiated in morphine (EC(50)=281 nM) compared to saline (EC(50)=8.8 microM) pretreated rats as indicated by a leftward shift in the concentration-response curve for met-enkephalin (ME)-induced currents. These currents were inhibited by superfusion of the mu-opioid receptor antagonist beta-funaltrexamine (beta-FNA) suggesting that repeated morphine administration enhances agonist stimulation of mu-opioid receptor coupling to G-proteins. Although supersensitivity of mu-opioid receptors in the PAG is counterintuitive to the development of tolerance, peak GIRK currents from tolerant rats desensitized more than currents from saline pretreated rats (56% of peak current after 10 min compared to 15%, respectively). These data indicate that antinociceptive tolerance may be triggered by enhanced agonist potency resulting in increased desensitization of mu-opioid receptors.

Evidence that calmodulin binding to the dopamine D2 receptor enhances receptor signaling.

The Ca2+ sensor calmodulin (CaM) regulates numerous proteins involved in G protein-coupled receptor (GPCR) signaling. CaM binds directly to some GPCRs, including the dopamine D2 receptor. We confirmed that the third intracellular loop of the D2 receptor is a direct contact point for CaM binding using coimmunoprecipitation and a polyHis pull-down assay, and we determined that the D2-like receptor agonist 7-OH-DPAT increased the colocalization of the D2 receptor and endogenous CaM in both 293 cells and in primary neostriatal cultures. The N-terminal three or four residues of D2-IC3 were required for the binding of CaM; mutation of three of these residues in the full-length receptor (I210C/K211C/I212C) decreased the coprecipitation of the D2 receptor and CaM and also significantly decreased D2 receptor signaling, without altering the coupling of the receptor to G proteins. Taken together, these findings suggest that binding of CaM to the dopamine D2 receptor enhances D2 receptor signaling.

Mu opioid receptor activation of ERK1/2 is GRK3 and arrestin dependent in striatal neurons.

In this study we investigated the mechanisms responsible for MAP kinase ERK1/2 activation following agonist activation of endogenous mu opioid receptors (MOR) normally expressed in cultured striatal neurons. Treatment with the MOR agonist fentanyl caused significant activation of ERK1/2 in neurons derived from wild type mice. Fentanyl effects were blocked by the opioid antagonist naloxone and were not evident in neurons derived from MOR knock-out (-/-) mice. In contrast, ERK1/2 activation by fentanyl was not evident in neurons from GRK3-/- mice or neurons pretreated with small inhibitory RNA for arrestin3. Consistent with this observation, treatment with the opiate morphine (which is less able to activate arrestin) did not elicit ERK1/2 activation in wild type neurons; however, transfection of arrestin3-(R170E) (a dominant positive form of arrestin that does not require receptor phosphorylation for activation) enabled morphine activation of ERK1/2. In addition, activation of ERK1/2 by fentanyl and morphine was rescued in GRK3-/- neurons following transfection with dominant positive arrestin3-(R170E). The activation of ERK1/2 appeared to be selective as p38 MAP kinase activation was not increased by either fentanyl or morphine treatment in neurons from wild type, MOR-/-, or GRK3-/- mice. In addition, U0126 (a selective inhibitor of MEK kinase responsible for ERK phosphorylation) blocked ERK1/2 activation by fentanyl. These results support the hypothesis that MOR activation of ERK1/2 requires opioid receptor phosphorylation by GRK3 and association of arrestin3 to initiate the cascade resulting in ERK1/2 phosphorylation in striatal neurons.

Kappa opioid receptor activation of p38 MAPK is GRK3- and arrestin-dependent in neurons and astrocytes.

AtT-20 cells expressing the wild-type kappa opioid receptor (KOR) increased phospho-p38 MAPK following treatment with the kappa agonist U50,488. The increase was blocked by the kappa antagonist norbinaltorphimine and not evident in untransfected cells. In contrast, U50,488 treatment of AtT-20 cells expressing KOR having alanine substituted for serine-369 (KSA) did not increase phospho-p38. Phosphorylation of serine 369 in the KOR carboxyl terminus by G-protein receptor kinase 3 (GRK3) was previously shown to be required for receptor desensitization, and the results suggest that p38 MAPK activation by KOR may require arrestin recruitment. This hypothesis was tested by transfecting arrestin3-(R170E), a dominant positive form of arrestin that does not require receptor phosphorylation for activation. AtT-20 cells expressing both KSA and arrestin3-(R170E) responded to U50,488 treatment with an increase in phospho-p38 consistent with the hypothesis. Primary cultured astrocytes (glial fibrillary acidic protein-positive) and neurons (gamma-aminobutyric acid-positive) isolated from mouse striata also responded to U50,488 by increasing phospho-p38 immunolabeling. p38 activation was not evident in either striatal astrocytes or neurons isolated from KOR knock-out mice or GRK3 knock-out mice. Astrocytes pretreated with small interfering RNA for arrestin3 were also unable to activate p38 in response to U50,488 treatment. Furthermore, in striatal neurons, the kappa-mediated phospho-p38 labeling was colocalized with arrestin3. These findings suggest that KOR may activate p38 MAPK in brain by a GRK3 and arrestin-dependent mechanism.

Dopamine D2 receptor stimulation of mitogen-activated protein kinases mediated by cell type-dependent transactivation of receptor tyrosine kinases.

Dopamine D2 receptor activation of extracellular signal-regulated kinases (ERKs) in non-neuronal human embryonic kidney 293 cells was dependent on transactivation of the platelet-derived growth factor (PDGF) receptor, as demonstrated by the effect of the PDGF receptor inhibitors tyrphostin A9 and AG 370 on quinpirole-induced phosphorylation of ERKs and by quinpirole-induced tyrosine phosphorylation of the PDGF receptor. In contrast, ectopically expressed D2 receptor or endogenous D2-like receptor activation of ERKs in NS20Y neuroblastoma cells, which express little or no PDGF receptor, or in rat neostriatal neurons was largely dependent on transactivation of the epidermal growth factor (EGF) receptor, as demonstrated using the EGF receptor inhibitor AG 1478 and by quinpirole-induced phosphorylation of the EGF receptor. The D2 receptor agonist quinpirole enhanced the coprecipitation of D2 and EGF receptors in NS20Y cells, suggesting that D2 receptor activation induced the formation of a macromolecular signaling complex that includes both receptors. Transactivation of the EGF receptor also involved the activity of a matrix metalloproteinase. Thus, although D2 receptor stimulation of ERKs in both cell lines was decreased by inhibitors of ERK kinase, Src-family protein tyrosine kinases, and serine/threonine protein kinases, D2-like receptors activated ERKs via transactivation of the EGF receptor in NS20Y neuroblastoma cells and rat embryonic neostriatal neurons, but via transactivation of the PDGF receptor in 293 cells.

Dopamine D1 receptor interaction with arrestin3 in neostriatal neurons.

Dopamine D1 receptor interactions with arrestins have been characterized using heterologously expressed D1 receptor and arrestins. The purpose of this study was to investigate the interaction of the endogenous D1 receptor with endogenous arrestin2 and 3 in neostriatal neurons. Endogenous arrestin2 and 3 in striatal homogenates bound to the C-terminus of the D1 receptor in a glutathione-S-transferase (GST) pulldown assay, with arrestin3 binding more strongly. The D1 C-terminus and, to a lesser extent, the third cytoplasmic loop also bound purified arrestin2 and 3. In neostriatal neurons, 2, 5, and 20 min agonist treatment increased the colocalization of the D1 receptor and arrestin3 immunoreactivity without altering the colocalization of the D1 receptor and arrestin2. Further, agonist treatment for 5 and 20 min caused translocation of arrestin3, but not arrestin2, to the membrane. The binding of arrestin3, but not arrestin2, to the D1 receptor was increased as assessed by coimmunoprecipitation after agonist treatment for 5 and 20 min. Agonist treatment of neurons induced D1 receptor internalization (35-45%) that was maximal within 2-5 min, a time-course similar to that of the increase in colocalization of the D1 receptor with arrestin3. These data indicate that the D1 receptor preferentially interacts with arrestin3 in neostriatal neurons.

Preferential Interaction between the dopamine D2 receptor and Arrestin2 in neostriatal neurons.

Dopamine D2 receptor interactions with arrestins and arrestin-dependent internalization have been characterized using heterologously expressed D2 receptor and arrestins. The purpose of this study was to investigate D2 receptor interaction with endogenous arrestins. Arrestin2 and arrestin3 in striatal homogenates bound to the third cytoplasmic loop of the D2 receptor, and purified arrestin2 and arrestin3 bound to the second and third loops and C terminus of the D2 receptor, in a glutathione S-transferase pull-down assay. In NS20Y neuroblastoma cells expressing an enhanced green-fluorescent protein-tagged D2 receptor (D2-EGFP), 2-h D2 agonist stimulation enhanced the colocalization of D2-EGFP with endogenous arrestin2 and arrestin3. These results suggest that the D2 receptor has the intrinsic ability to bind both nonvisual arrestins. Agonist treatment of D2-EGFP NS20Y cells induced D2 receptor internalization (36-46%) that was maximal within 20 min, but that was prevented by small interfering RNA-induced depletion of arrestin2 and arrestin3. In neostriatal neurons, 2-h agonist treatment selectively increased the colocalization of the endogenous D2 receptor with arrestin2, whereas receptor colocalization with arrestin3 was reduced. Agonist stimulation caused translocation of arrestin2, but not arrestin3, to the membrane in neurons and selectively enhanced the coimmunoprecipitation of the D2 receptor and arrestin2. All three measures of receptor/arrestin interaction (colocalization, translocation, and coprecipitation) demonstrated selective agonist-induced interaction between the D2 receptor and arrestin2 in neurons.