Validation of a molecular diagnostic assay for CALR exon 9 indels in myeloproliferative neoplasms: identification of coexisting JAK2 and CALR mutations and a novel 9 bp deletion in CALR.

INTRODUCTION: The 2008 WHO criteria for the diagnosis and classification of myeloproliferative neoplasms (MPN) rely in part upon the assessment of mutations in JAK2 and MPL genes. Recently, mutations in calreticulin (CALR) have been identified in MPN lacking JAK2 and MPL mutations. We have validated a sensitive fragment analysis assay to detect CALR mutations. METHODS: Genomic DNA from peripheral blood, bone marrow, and FFPE bone marrow clot preparations from 52 MPN specimens with known JAK2 and MPL mutation status and 29 non-MPN specimens was analyzed. CALR mutation testing was performed by fragment length analysis, and the results were confirmed by sequencing. Accuracy, precision, sensitivity, specificity, and robustness of the assay were determined. RESULTS: Forty specimens (32 JAK2+, 2 JAK2-/MPL+, and 6 JAK2-/MPL-) were negative for CALR mutations. Twelve specimens had CALR mutations including 52 bp deletion (5), 5 bp insertion (6), and a novel 9 bp deletion (1). This 9 bp inframe deletion occurring at an allelic burden of 50% would delete three amino acids. One specimen with a 52 bp deletion also had JAK2 V617F mutation. All 29 non-MPN specimens were negative for CALR mutations. The assay accurately identified the mutation status of all 52 MPN specimens and had a coefficient of variation <3% for the fragment size and mutant peaks with a sensitivity of 5% for indels. CONCLUSIONS: Fragment analysis is an accurate and sensitive method for the detection of CALR indels. The novel 9 bp deletion is likely a germline variant. Consequence of coexisting JAK2 V617F and CALR mutations requires careful interpretation.

Loss of signalling via Gα13 in germinal centre B-cell-derived lymphoma.

Germinal centre B-cell-like diffuse large B-cell lymphoma (GCB-DLBCL) is a common malignancy, yet the signalling pathways that are deregulated and the factors leading to its systemic dissemination are poorly defined. Work in mice showed that sphingosine-1-phosphate receptor-2 (S1PR2), a Gα12 and Gα13 coupled receptor, promotes growth regulation and local confinement of germinal centre B cells. Recent deep sequencing studies of GCB-DLBCL have revealed mutations in many genes in this cancer, including in GNA13 (encoding Gα13) and S1PR2 (refs 5,6, 7). Here we show, using in vitro and in vivo assays, that GCB-DLBCL-associated mutations occurring in S1PR2 frequently disrupt the receptor's Akt and migration inhibitory functions. Gα13-deficient mouse germinal centre B cells and human GCB-DLBCL cells were unable to suppress pAkt and migration in response to S1P, and Gα13-deficient mice developed germinal centre B-cell-derived lymphoma. Germinal centre B cells, unlike most lymphocytes, are tightly confined in lymphoid organs and do not recirculate. Remarkably, deficiency in Gα13, but not S1PR2, led to germinal centre B-cell dissemination into lymph and blood. GCB-DLBCL cell lines frequently carried mutations in the Gα13 effector ARHGEF1, and Arhgef1 deficiency also led to germinal centre B-cell dissemination. The incomplete phenocopy of Gα13- and S1PR2 deficiency led us to discover that P2RY8, an orphan receptor that is mutated in GCB-DLBCL and another germinal centre B-cell-derived malignancy, Burkitt's lymphoma, also represses germinal centre B-cell growth and promotes confinement via Gα13. These findings identify a Gα13-dependent pathway that exerts dual actions in suppressing growth and blocking dissemination of germinal centre B cells that is frequently disrupted in germinal centre B-cell-derived lymphoma.

Oncogenically active MYD88 mutations in human lymphoma.

The activated B-cell-like (ABC) subtype of diffuse large B-cell lymphoma (DLBCL) remains the least curable form of this malignancy despite recent advances in therapy. Constitutive nuclear factor (NF)-κB and JAK kinase signalling promotes malignant cell survival in these lymphomas, but the genetic basis for this signalling is incompletely understood. Here we describe the dependence of ABC DLBCLs on MYD88, an adaptor protein that mediates toll and interleukin (IL)-1 receptor signalling, and the discovery of highly recurrent oncogenic mutations affecting MYD88 in ABC DLBCL tumours. RNA interference screening revealed that MYD88 and the associated kinases IRAK1 and IRAK4 are essential for ABC DLBCL survival. High-throughput RNA resequencing uncovered MYD88 mutations in ABC DLBCL lines. Notably, 29% of ABC DLBCL tumours harboured the same amino acid substitution, L265P, in the MYD88 Toll/IL-1 receptor (TIR) domain at an evolutionarily invariant residue in its hydrophobic core. This mutation was rare or absent in other DLBCL subtypes and Burkitt's lymphoma, but was observed in 9% of mucosa-associated lymphoid tissue lymphomas. At a lower frequency, additional mutations were observed in the MYD88 TIR domain, occurring in both the ABC and germinal centre B-cell-like (GCB) DLBCL subtypes. Survival of ABC DLBCL cells bearing the L265P mutation was sustained by the mutant but not the wild-type MYD88 isoform, demonstrating that L265P is a gain-of-function driver mutation. The L265P mutant promoted cell survival by spontaneously assembling a protein complex containing IRAK1 and IRAK4, leading to IRAK4 kinase activity, IRAK1 phosphorylation, NF-κB signalling, JAK kinase activation of STAT3, and secretion of IL-6, IL-10 and interferon-ß. Hence, the MYD88 signalling pathway is integral to the pathogenesis of ABC DLBCL, supporting the development of inhibitors of IRAK4 kinase and other components of this pathway for the treatment of tumours bearing oncogenic MYD88 mutations.

Chronic active B-cell-receptor signalling in diffuse large B-cell lymphoma.

A role for B-cell-receptor (BCR) signalling in lymphomagenesis has been inferred by studying immunoglobulin genes in human lymphomas and by engineering mouse models, but genetic and functional evidence for its oncogenic role in human lymphomas is needed. Here we describe a form of 'chronic active' BCR signalling that is required for cell survival in the activated B-cell-like (ABC) subtype of diffuse large B-cell lymphoma (DLBCL). The signalling adaptor CARD11 is required for constitutive NF-kappaB pathway activity and survival in ABC DLBCL. Roughly 10% of ABC DLBCLs have mutant CARD11 isoforms that activate NF-kappaB, but the mechanism that engages wild-type CARD11 in other ABC DLBCLs was unknown. An RNA interference genetic screen revealed that a BCR signalling component, Bruton's tyrosine kinase, is essential for the survival of ABC DLBCLs with wild-type CARD11. In addition, knockdown of proximal BCR subunits (IgM, Ig-kappa, CD79A and CD79B) killed ABC DLBCLs with wild-type CARD11 but not other lymphomas. The BCRs in these ABC DLBCLs formed prominent clusters in the plasma membrane with low diffusion, similarly to BCRs in antigen-stimulated normal B cells. Somatic mutations affecting the immunoreceptor tyrosine-based activation motif (ITAM) signalling modules of CD79B and CD79A were detected frequently in ABC DLBCL biopsy samples but rarely in other DLBCLs and never in Burkitt's lymphoma or mucosa-associated lymphoid tissue lymphoma. In 18% of ABC DLBCLs, one functionally critical residue of CD79B, the first ITAM tyrosine, was mutated. These mutations increased surface BCR expression and attenuated Lyn kinase, a feedback inhibitor of BCR signalling. These findings establish chronic active BCR signalling as a new pathogenetic mechanism in ABC DLBCL, suggesting several therapeutic strategies.

Prognostic value of regulatory T cells, lymphoma-associated macrophages, and MUM-1 expression in follicular lymphoma treated before and after the introduction of monoclonal antibody therapy: a Southwest Oncology Group Study.

BACKGROUND: The purpose was to examine the prognostic impact of features of tumor cells and immune microenvironment in patients with follicular lymphoma treated with and without anti-CD20 monoclonal antibody therapy. PATIENTS AND METHODS: Tissue microarrays were constructed from archived tissue obtained from patients on three sequential Southwest Oncology Group (SWOG) trials for FL. All three trials included anthracycline-based chemotherapy. Anti-CD20 monoclonal antibodies were included for patients in the latter two trials. Immunohistochemistry was used to study the number and distribution of cells staining for forkhead box protein P3 (FOXP3) and lymphoma-associated macrophages (LAMs) and the number of lymphoma cells staining for myeloma-associated antigen-1 (MUM-1). Cox proportional hazards regression was used to evaluate the association between marker expression and overall survival (OS). RESULTS: The number or pattern of infiltrating FOXP3 cells and LAMs did not correlate with OS in sequential SWOG studies for FL. The presence of MUM-1 correlated with lower OS for patients who received monoclonal antibody but not for those treated with chemotherapy alone. CONCLUSIONS: Immune cell composition of lymph nodes did not correlate with OS in this analysis of trials in FL. The mechanism of the observed correlation between MUM-1 expression and adverse prognosis in patients receiving monoclonal antibody therapy requires confirmation.

Stromal gene signatures in large-B-cell lymphomas.

BACKGROUND: The addition of rituximab to combination chemotherapy with cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP), or R-CHOP, has significantly improved the survival of patients with diffuse large-B-cell lymphoma. Whether gene-expression signatures correlate with survival after treatment of diffuse large-B-cell lymphoma is unclear. METHODS: We profiled gene expression in pretreatment biopsy specimens from 181 patients with diffuse large-B-cell lymphoma who received CHOP and 233 patients with this disease who received R-CHOP. A multivariate gene-expression-based survival-predictor model derived from a training group was tested in a validation group. RESULTS: A multivariate model created from three gene-expression signatures--termed "germinal-center B-cell," "stromal-1," and "stromal-2"--predicted survival both in patients who received CHOP and patients who received R-CHOP. The prognostically favorable stromal-1 signature reflected extracellular-matrix deposition and histiocytic infiltration. By contrast, the prognostically unfavorable stromal-2 signature reflected tumor blood-vessel density. CONCLUSIONS: Survival after treatment of diffuse large-B-cell lymphoma is influenced by differences in immune cells, fibrosis, and angiogenesis in the tumor microenvironment.

hTid-1, a human DnaJ protein, modulates the interferon signaling pathway.

The Jak family of protein-tyrosine kinases are crucial for the signaling of a large number of different polypeptide ligands, including the interferons, many cytokines, erythropoietin, and growth factors. Through their interaction with receptors, the Jaks initiate a signaling cascade resulting in the activation of gene transcription and ultimately a cellular response to various ligands. In addition to their role in cellular signaling, alteration of Jak activity has been implicated in several disease states. In identifying Jak2-interacting proteins with the yeast two-hybrid system, we cloned the human homologue of the Drosophila melanogaster tumor suppressor gene lethal () tumorous imaginal discs, which encodes the protein Tid56. Drosophila Tid56 and its human homologue hTid-1 represent members of the DnaJ family of molecular chaperones. The TID1 gene encodes two splice variants hTid-1(S) and hTid-1(L). We confirmed the interaction between Jak2 and hTid-1(S) or hTid-1(L) by immunoprecipitation from COS-1 cells expressing these proteins. The interaction between endogenous hTid-1 and Jak2 was shown in HEp2 cells. We further showed that hTid-1 interacts with the human interferon-gamma (Hu-IFN-gamma) receptor subunit IFN-gamma R2. In addition, using a chimeric construct where the extracellular domain of IFN-gamma R2 was fused to the kinase domain of Jak2, we showed that hTid-1 binds more efficiently to the chimera with an active kinase domain than to a similar construct with an inactive kinase domain. Additionally, the data demonstrate that hTid-1 isoforms as well as Jak2 interact with Hsp70/Hsc70 in vivo, and the interaction between Hsp70/Hsc70 and hTid-1 is reduced after IFN-gamma treatment. Furthermore, both hTid-1(S) and hTid-1(L) can modulate IFN-gamma-mediated transcriptional activity.

Anaplastic lymphoma kinase (ALK) expression in the inflammatory myofibroblastic tumor: a comparative immunohistochemical study.

Inflammatory myofibroblastic tumor (IMT) is an uncommon mesenchymal neoplasm with a variable histologic appearance that may mimic other spindle cell processes, particularly nodular fasciitis, desmoid tumor, and in intra-abdominal locations, gastrointestinal stromal tumor. Recently, gene fusions involving ALK at chromosome 2p23 have been described in IMTs. The resultant ALK protein overexpression in the myofibroblastic component of these tumors is detectable by immunohistochemistry. We examined 73 IMTs, 20 cases of nodular fasciitis, 15 desmoid fibromatoses, and 15 gastrointestinal stromal tumors by immunohistochemistry using ALK-11, a rabbit polyclonal antibody that recognizes the C-terminus of the protein. ALK positivity was detected in 44 of 73 (60%) IMTs. All cases of nodular fasciitis, desmoid fibromatosis, and gastrointestinal stromal tumors were ALK negative (p < 0.001). These findings demonstrate that ALK positivity is common in IMTs, and immunohistochemistry using anti-ALK antibodies can be helpful in the differential diagnosis of these neoplasms. In addition, anti-ALK staining seems to correlate with those IMTs that have the typical tri-patterned histologic appearance and clinical presentation, providing additional support to the premise that IMT is a distinctive clinicopathologic entity within the broad category of inflammatory pseudotumors.

Effectiveness of bi-atrial pacing for reducing atrial fibrillation after coronary artery bypass graft surgery.

Atrial fibrillation (AF) is common after cardiac surgery and adds significant cost and morbidity. The use of prophylactic pacing strategies to prevent post-operative AF has been controversial. We previously performed a pilot study which suggested that the combination of beta-blockers and bi-atrial pacing (BAP) may reduce AF after cardiac surgery. We prospectively randomized 118 patients to continuous BAP for up to 96 hours post-operatively versus standard therapy. All patients were treated with beta-blockers as tolerated. Patients were paced in the AAI mode at a rate of 100 pulses per minute. The primary endpoint of the study was the occurrence of sustained AF (>10 minutes). There was a significant reduction in the incidence of AF in the BAP group among patients undergoing coronary artery bypass graft surgery with or without aortic valve replacement (35 % vs. 19 % AF; OR=0.38, 95 % CI 0.15, 0.93; p <0.05). Including patients undergoing isolated aortic valve surgery (n=7), there remained a strong trend toward a reduction of AF with pacing (no atrial pacing [NAP] vs. BAP; 35 % vs. 21 % AF; OR=0.48, 95 % CI 0.21, 1.11; p=0.08). Patients age 70 or greater benefited most from pacing (NAP vs. BAP; 55 vs. 25 % AF; p<0.05), while those less than 70 years of age did not (17 vs. 18 % p=NS). There was a significant reduction in the amount of time spent in the intensive care unit among patients receiving BAP (50+/-40 vs. 37+/-25 h; p<0.05).BAP together with beta-blockade after coronary artery bypass graft surgery reduces the incidence of post-operative atrial AF. Elderly patients (age 70 or greater) appear to benefit most, and may be a group to whom this therapy should be targeted.

Induction of the ubiquitin-proteasome pathway during the keratocyte transition to the repair fibroblast phenotype.

PURPOSE: To examine dynamics and function of the ubiquitin (Ub)-proteasome pathway (UPP) during corneal stromal cell acquisition of the repair fibroblast phenotype. METHODS: An established cell culture model was used in which freshly isolated rabbit corneal stromal cells acquire a repair fibroblast phenotype, thereby mimicking injury-induced stromal cell activation. RESULTS: Transition to the repair fibroblast phenotype during the 72 hours after initial plating was coincident with progressive UPP induction. Levels of Ub, Ub-conjugated proteins, ubiquitinylating enzymes E1 and E2-25K, and 26 S proteasome increased two- to fivefold in activated stromal cells. These increases were associated with enhanced (>10-fold) capacity for Ub-dependent proteolysis of (125)I-labeled H2A and with progressive (>6-fold) increases in the UPP substrate, inhibitor of kappaBalpha (IkappaBalpha). Because IkappaBalpha expression is induced by nuclear factor (NF)-kappaB, this finding suggests that rates of constitutive NF-kappaB activation, and thus IkappaBalpha degradation, are elevated in activated stromal cells. Both freshly isolated and activated stromal cells degraded IkappaBalpha in response to IL-1alpha; yet, only activated stromal cells maintained autocrine IL-1alpha expression after 24 hours. UPP induction was coincident with a more than 90% loss of tissue transketolase (TKT) and aldehyde dehydrogenase (ALDH) class 1. TKT was stabilized during the repair phenotype transition by proteasome inhibition and was degraded (>30%/h) by the UPP in cell-free assays. CONCLUSIONS: Coordinate induction of the UPP during stromal cell activation alters levels of IkappaBalpha and TKT, two UPP substrates that are implicated in the loss of tissue stasis and corneal clarity after injury.

PRMT5 (Janus kinase-binding protein 1) catalyzes the formation of symmetric dimethylarginine residues in proteins.

We have identified a new mammalian protein arginine N-methyltransferase, PRMT5, formerly designated Janus kinase-binding protein 1, that can catalyze the formation of omega-N(G)-monomethylarginine and symmetric omega-N(G),N(G')-dimethylarginine in a variety of proteins. A hemagglutinin peptide-tagged PRMT5 complex purified from human HeLa cells catalyzes the S-adenosyl-l-[methyl-(3)H]methionine-dependent in vitro methylation of myelin basic protein. When the radiolabeled myelin basic protein was acid-hydrolyzed to free amino acids, and the products were separated by high-resolution cation exchange chromatography, we were able to detect two tritiated species. One species co-migrated with a omega-N(G)-monomethylarginine standard, and the other co-chromatographed with a symmetric omega-N(G),N(G')-dimethylarginine standard. Upon base treatment, this second species formed methylamine, a breakdown product characteristic of symmetric omega-N(G),N(G')-dimethylarginine. Further analysis of these two species by thin layer chromatography confirmed their identification as omega-N(G)-monomethylarginine and symmetric omega-N(G),N(G')-dimethylarginine. The hemagglutinin-PRMT5 complex was also able to monomethylate and symmetrically dimethylate bovine histone H2A and a glutathione S-transferase-fibrillarin (amino acids 1-148) fusion protein (glutathione S-transferase-GAR). A mutation introduced into the S-adenosyl-l-methionine-binding motif I of a myc-tagged PRMT5 construct in COS-1 cells led to a near complete loss of observed enzymatic activity. PRMT5 is the first example of a catalytic chain for a type II protein arginine N-methyltransferase that can result in the formation of symmetric dimethylarginine residues as observed previously in myelin basic protein, Sm small nuclear ribonucleoproteins, and other polypeptides.

Trefoil peptides promote restitution of wounded corneal epithelial cells.

The ocular surface shares many characteristics with mucosal surfaces. In both, healing is regulated by peptide growth factors, cytokines, and extracellular matrix proteins. However, these factors are not sufficient to ensure most rapid healing. Trefoil peptides are abundantly expressed epithelial cell products which exert protective effects and are key regulators of gastrointestinal epithelial restitution, the critical early phase of cell migration after mucosal injury. To assess the role of trefoil peptides in corneal epithelial wound healing, the effects of intestinal trefoil factor (ITF/TFF3) and spasmolytic polypeptide (SP/TFF2) on migration and proliferation of corneal epithelial cells were analyzed. Both ITF and SP enhanced restitution of primary rabbit corneal epithelial cells in vitro. While the restitution-enhancing effects of TGF-alpha and TGF-beta were both inhibited by neutralizing anti-TGF-beta-antibodies, trefoil peptide stimulation of restitution was not. Neither trefoil peptide significantly affected proliferation of primary corneal epithelial cells. ITF but not SP or pS2 mRNA was present in rabbit corneal and conjunctival tissues. In summary, the data indicate an unanticipated role of trefoil peptides in healing of ocular surface and demand rating their functional actions beyond the gastrointestinal tract.

Effect of lovastatin on cardiovascular resource utilization and costs in the Air Force/Texas Coronary Atherosclerosis Prevention Study (AFCAPS/TexCAPS). AFCAPS/TexCAPS Research Group.

This cost-consequences analysis of the Air Force/Texas Coronary Atherosclerosis Prevention Study compares the costs of lovastatin treatment with the costs of cardiovascular hospitalizations and procedures. The cost of lovastatin treatment was defined as the average retail price and the cost of drug safety monitoring and adverse experiences. Costs were determined by actual rates of hospitalizations and procedures. Within a trial, lovastatin treatment cost approximately $4,654/patient. Lovastatin treatment significantly reduced the cumulative rate of cardiovascular hospitalizations and procedures (p = 0.002). Over the duration of the study, the cumulative number of cardiovascular hospitalizations and related therapeutic procedures was significantly reduced by 29%. The time to first cardiovascular-related hospitalization or procedure was significantly extended by lovastatin (p = 0.002). Lovastatin reduced the frequency of cardiovascular hospitalization (28%), and cardiovascular therapeutic (32%) and diagnostic procedures (23%). Among therapeutic procedures, treatment reduced coronary artery bypass graft surgery by 19% and percutaneous transluminal coronary angioplasty by 37%. Total cardiovascular-related hospital days were reduced by 26% (p = 0.025). The between-group offset in direct medical costs was $524, which resulted in a 11% cost offset of lovastatin therapy over the mean study duration of 5.2 years. Lovastatin provides meaningful reductions in cardiovascular-related resource utilization and reductions in direct cardiovascular-related costs associated with the onset of coronary disease.

Does cardioplegia type affect outcome and survival in patients with advanced left ventricular dysfunction? Results from the CABG Patch Trial.

BACKGROUND: There is controversy regarding which cardioplegic solution, temperature, and route of administration provides superior protection. The CABG Patch Trial enrolled a high-risk group of coronary artery disease patients with an ejection fraction of <36%. Thus, they constitute an ideal group to benefit most from optimal cardioplegic protection. METHODS AND RESULTS: All patients randomized into the trial were compared with respect to the use of blood and crystalloid cardioplegia. In addition, a questionnaire was sent to surgeons requesting blood cardioplegic temperature and route. Patients receiving crystalloid cardioplegia versus those receiving blood cardioplegia were found to have significantly more operative deaths (2% versus 0.3%, P:=0.02), postoperative myocardial infarctions (10% versus 2%, P:<0.001), shock (13% versus 7%, P:=0. 013), and postoperative conduction defects (21.6% versus 12.4%, P:=0. 001). Despite this, early death (6% crystalloid versus 4% blood cardioplegia) and late death (24% crystalloid versus 21% blood cardioplegia) statistics were not significantly different. Patients receiving normothermic blood had less postoperative right ventricular dysfunction (10%) than did patients receiving cold blood (25%) or cold blood with warm reperfusion (30%) (P:=0.004). There was no significant difference in early or late death. Finally, patients who received combined antegrade and retrograde cardioplegia had significantly less inotrope use (71% versus 84%, P:=0.002), right ventricular dysfunction (23% versus 41%, P:=0.001), and postoperative balloon pump use (12% versus 19%, P:=0.02) than did those who received antegrade cardioplegia. There was no difference in survival. CONCLUSIONS: Blood cardioplegia and combined antegrade and retrograde cardioplegia are superior to crystalloid and antegrade cardioplegia alone for postoperative morbidity. Despite this, there is no significant difference in early or late survival.

Squamous cell carcinoma arising in recurrent respiratory papillomatosis with pulmonary involvement: emerging common pattern of clinical features and human papillomavirus serotype association.

Squamous papillomas of the lung are an uncommon feature of recurrent respiratory papillomatosis, occurring in fewer than 1% of cases. We describe a 23-year-old patient with pulmonary papillomas who developed a fatal squamous cell carcinoma of the lung. PCR-based human papillomavirus (HPV) typing showed the presence of HPV 11 DNA in both benign papillomas and invasive carcinoma. A review of the literature reveals four reports of malignant transformation of juvenile-onset recurrent respiratory papillomatosis in which HPV typing was performed. Similar clinical features are noted in all of the reports; specifically, each case has arisen in a young adult man with a history of papillomatosis since childhood. In each of the cases, HPV 11 was identified in association with the squamous cell carcinoma. Although HPV 11 is uncommonly associated with the development of invasive carcinoma at other sites, these findings suggest that it is correlated with malignant transformation in the setting of juvenile-onset recurrent respiratory papillomatosis.

Hsl7p, the yeast homologue of human JBP1, is a protein methyltransferase.

The yeast protein Hsl7p is a homologue of Janus kinase binding protein 1, JBP1, a newly characterized protein methyltransferase. In this report, Hsl7p also is shown to be a methyltransferase. It can be crosslinked to [(3)H]S-adenosylmethionine and exhibits in vitro protein methylation activity. Calf histones H2A and H4 and bovine myelin basic protein were methylated by Hsl7p, whereas histones H1, H2B, and H3 and bovine cytochrome c were not. We demonstrated that JBP1 can complement Saccharomyces cerevisiae with a disrupted HSL7 gene as judged by a reduction of the elongated bud phenotype, and a point mutation in the JBP1 S-adenosylmethionine consensus binding sequence eliminated all complementation by JBP1. Therefore, we conclude the yeast protein Hsl7p is a sequence and functional homologue of JBP1. These data provide evidence for an intricate link between protein methylation and macroscopic changes in yeast morphology.

Determinants of outcome in patients with sustained ventricular tachyarrhythmias: the antiarrhythmics versus implantable defibrillators (AVID) study registry.

BACKGROUND: The prognosis of patients with sustained ventricular tachyarrhythmias varies according to clinical characteristics. We sought to identify predictors of survival in a large population of patients with documented sustained ventricular tachyarrhythmias not related to reversible or correctable causes included in the Antiarrhythmics Versus Implantable Defibrillators (AVID) Registry. METHODS AND RESULTS: We analyzed the impact of 36 demographic, clinical, and discharge treatment variables on the outcome for 3559 patients. Survival status was assessed with the use of the National Death Index. Multivariate analyses were performed with the use of the Cox proportional hazards model. After a mean follow-up of 17 +/- 12 months, 631 patients died. Actuarial survival was 0.86 (95% confidence interval [CI] 0.85 to 0.88), 0.79 (95% CI 0.78 to 0.81), and 0.72 (95% CI 0.70 to 0.74) at 1, 2, and 3 years. Multivariate predictors of worse survival included older age, severe left ventricular dysfunction, lower systolic blood pressure, history of congestive heart failure, diabetes, smoking or atrial fibrillation, and preexistent pacemaker. The hemodynamic impact of the qualifying arrhythmia was not a predictor of outcome. Defibrillator implantation and hospital discharge while the patient was taking a beta-blocker or an angiotensin-converting enzyme inhibitor were associated with better prognosis. CONCLUSIONS: Despite therapeutic advances, the mortality rates of patients with sustained ventricular tachyarrhythmias remain high. Prognosis depends on the severity of underlying heart disease, as reflected by the extent of left ventricular dysfunction and the presence of heart failure. Well-tolerated ventricular tachycardia in patients with structural heart disease does not carry a significantly better prognosis than ventricular tachyarrhythmia with more severe hemodynamic consequences.

Effects of adenosine on local stimulus-response latency and induction of atrial fibrillation by premature stimuli.

Premature atrial stimuli delivered during the relative refractory or "vulnerable" period exhibit increased local stimulus-response latency and may occasionally induce atrial arrhythmias. The use of adenosine to treat supraventricular tachycardias may also provoke atrial arrhythmias. In this study we investigated the effects of adenosine on the latency of premature complexes in relation to repolarization and induction of atrial arrhythmias in 14 patients without structural heart disease. A monophasic action potential catheter was used for recording in the right atrium and introducing premature stimuli (S2) at twice diastolic threshold after eight paced (S1) complexes. At short coupling intervals, S2 latency increased relative to S1 latency. S2 was delivered repeatedly at a fixed coupling interval (producing maximal local response latency) and adenosine (6 mg) was given intravenously. Adenosine decreased S2 latency significantly (23+/-5 to 11+/-3 ms, P<0.01), to values similar to S, latency. However, despite the decrease in S2 latency, the combination of adenosine and S2 more often resulted in transient atrial arrhythmias (11 of 14 patients vs 2 of 14 patients without adenosine, P<0.05). Adenosine had no effect on S, latency (9+/-2 vs. 9+/-2 ms) but decreased monophasic action potential duration from 202+/-37 to 158+/-38 ms (P<0.01). Adenosine was also given to 10 patients with S2 introduced at a coupling interval 40-50 ms less than the baseline effective refractory period. This resulted in a decrease in atrial refractoriness and capture of S2 in all cases. Latency for S2 was significantly greater than Si latency (21+/-12 vs. 9+/-2 ms, P<0.01) and transient atrial arrhythmias were induced in 9 of 10 patients. We conclude that for a given S2 coupling interval, adenosine decreases local stimulus-response latency but increases atrial vulnerability to transient atrial arrhythmias. Decreased latency may be related to a shift in the zone of relative refractoriness associated with an adenosine-mediated decrease in monophasic action potential duration. Induction of atrial arrhythmias in the presence of adenosine occurs independently of increased latency and is therefore not dependent on S2 falling within the relative refractory period at the site of stimulation.

Economic impact of GPIIB/IIIA blockade after high-risk angioplasty: results from the RESTORE trial. Randomized Efficacy Study of Tirofiban for Outcomes and Restenosis.

OBJECTIVE: This study was conducted to assess the impact of GPIIb/IIIa blockade with tirofiban on costs during the initial hospitalization and at 30 days among patients undergoing high-risk coronary angioplasty. BACKGROUND: GPIIb/IIIa blockers are a new class of compounds that have been shown in clinical studies to prevent complications after high-risk angioplasty. METHODS: The RESTORE trial was a multinational, blinded placebo-controlled study of 2,197 patients randomized to tirofiban or placebo following coronary angioplasty. This economic assessment was a prospective substudy of the RESTORE trial, and included 1,920 patients enrolled in the U.S. Costs were estimated for the U.S. cohort based on their utilization of healthcare resources and on costs measured directly in 820 U.S. patients at 30 sites. RESULTS: There was a 36% difference in the rate of the composite event of death, myocardial infarction (MI) and revascularization at two days between tirofiban and placebo (8% vs. 12%, p = 0.002). This difference was attributed to a reduction in nonfatal MI, repeat angioplasty, coronary surgery and stent placement. These clinical benefits followed a similar trend at 30 days, with a 16% reduction in the composite event (p = 0.10). In-hospital cost, including professional and study drug costs, was $12,145 +/- 5,882 with placebo versus $12,230 +/- 5,527 with tirofiban (p = 0.75). The 30-day cost was $12,402 +/- 6,147 with placebo versus $12,446 +/- 5,814 with tirofiban (p = 0.87). CONCLUSIONS: Tirofiban has been shown to decrease in-hospital and possibly 30-day events after high-risk angioplasty. The beneficial clinical effects of tirofiban in high-risk patients can be achieved at no increased cost.

Presence of angiographic coronary collaterals predicts myocardial recovery after coronary bypass surgery in patients with severe left ventricular dysfunction.

BACKGROUND: Patients with coronary artery disease and left ventricular dysfunction (LVD) may have areas of hibernating myocardium that improve functionally after revascularization. Coronary collateral circulation may sustain ischemic, dysfunctional myocardium and favor myocardial recovery after revascularization. We evaluated the effect of angiographic coronary collaterals on myocardial functional recovery after coronary bypass graft (CABG) surgery in a group of patients with severe LVD. METHODS AND RESULTS: Forty-one patients with multivessel coronary artery disease and advanced LVD (left ventricular ejection fraction [LVEF] 25 +/- 5%) undergoing CABG were identified from a prospective database. Preoperative coronary angiograms were evaluated for collaterals, which were graded according to Rentrop's classification (0 to 3), and a collateral index was calculated (collateral sum divided by 3). Preoperative and postoperative radionuclide ventriculograms provided global LVEF and regional ejection fractions. Of 123 regions evaluated, 120 were dysfunctional at baseline. Virtually all (122 of 123) regions were subtended by an artery with > or = 70% stenosis that was bypassed. Thirty-eight (81%) of 47 dysfunctional regions with grade 2 or 3 collaterals improved regional ejection fraction after surgery versus 38 (52%) of 73 dysfunctional regions with grade 0 or 1 collaterals (P = 0.0018). Global LVEF was 34 +/- 10% after surgery (P < 0.001 versus before surgery). Among patients with a global LVEF increase > or = 10%, collateral index was 1.81 versus 0.83 in those with an LVEF increase < 10% (P = 0.005). CONCLUSIONS: In this population of patients with coronary artery disease with severe LVD, the presence of angiographic grade 2 or 3 collaterals predicted recovery of regional and global myocardial function after CABG.