Venous thromboembolism in patients with pancreatic adenocarcinoma: Disease burden and initiation of ambulatory thromboprophylaxis.

BACKGROUND/OBJECTIVES: Ambulatory thromboprophylaxis (AT) in patients with pancreatic adenocarcinoma (PAC) reduces venous thromboembolism (VTE) risk and is recommended for patients receiving systemic chemotherapy. We evaluated VTE rates, severity, timing, and risk factors in PAC patients as well as AT rates and initiation times. METHODS: Patients diagnosed with PAC were included. Data collected included patient demographics, medical history, PAC diagnosis, development of VTE, AT, and bleeding episodes. VTE was defined as a DVT or a PE. Patients were classified as receiving AT for VTE prevention if they received a prescription for outpatient anticoagulation. RESULTS: The cohort included 243 PAC patients. VTE occurred in 24 %. Overall, 52 % developing VTE were hospitalized and 5 % died as a result of the VTE. Of those who developed VTE 50 % were diagnosed within the first 2 months of PAC diagnosis. Univariate predictors of elevated VTE risk included an elevated Onkotev score, metastasis at diagnosis, male gender and not receiving AT. Multivariate predictors of elevated VTE risk included male gender (P = 0.014) and not receiving AT (P = 0.001). Overall, 30 % of patients received AT. The median time from diagnosis to initiation of AT was 43 days. Major bleeding occurred in 5.8 %. Patients receiving AT were not at a significantly increased risk of major bleeding (p = 0.5). Patients with intestinal tumor invasion were at significantly increased risk of major bleeding (P = 0.021). CONCLUSION: VTE risk is significant and morbid in PAC patients. AT rates are low, and initiation is often delayed. Therapeutic endoscopists diagnosing PAC may be helpful in AT initiation.

Effect of rosuvastatin on risk markers for venous thromboembolism in cancer.

Essentials Statins lower venous thromboembolism risk in general but have not been studied in cancer patients. We completed a randomized trial of rosuvastatin vs. placebo among cancer patients on chemotherapy. Rosuvastatin did not significantly lower prothrombotic biomarkers including D-dimer. The role of statins in venous thrombosis prevention in cancer patients remains unknown. SUMMARY: Background Statin therapy is associated with lower risk of venous thromboembolism (VTE) but has not been prospectively evaluated in patients with advanced cancer. Objectives We determined if statin administration in this high-risk population reduces the risk of VTE, based on established and emerging biomarkers. Patients/Methods This double-blind, crossover, randomized controlled trial among patients with advanced cancer receiving systemic therapy allocated participants to rosuvastatin 20 mg daily or placebo for 3-4 weeks prior to crossover to the alternative therapy, with a 3-5-week washout. D-dimer, C-reactive protein (CRP), soluble (s)P-selectin, factor VIII (FVIII), thrombin generation and exploratory biomarkers focusing on endogenous thrombin potential, including tissue factor (TF), activated factor IX (FIXa) and activated factor XI (FXIa), were measured at the start and end of both treatment periods. The primary outcome was change in D-dimer with rosuvastatin compared with placebo. Results Of 38 enrolled participants, 24 (63%) completed the study. Rosuvastatin did not cause statistically significant changes in D-dimer levels or any other biomarker. CRP levels decreased by 40%; 4.3 mg L-1 (95% confidence interval, -11.0 to +2.5 mg L-1 ) compared with placebo. In post-hoc analysis, participants who received rosuvastatin initially during their first line of treatment had a 13% decrease in D-dimer. Circulating TF, FIXa and FXIa were detected in 26%, 68% and 71% of cancer patients despite not being found in healthy individuals. Conclusions Rosuvastatin did not cause favorable changes in biomarkers of VTE risk in advanced cancer patients receiving chemotherapy. The role of statin therapy as thromboprophylaxis in the cancer population remains uncertain.

Randomized phase II study of loratadine for the prevention of bone pain caused by pegfilgrastim.

PURPOSE: Bone pain is a common side effect of pegfilgrastim and can interfere with quality of life and treatment adherence. This study investigated the impact of antihistamine prophylaxis on pegfilgrastim-induced bone pain. METHODS: This is a two-stage enrichment trial design. Patients receiving an initial dose of pegfilgrastim after chemotherapy were enrolled into the observation (OBS) stage. Those who developed significant back or leg bone pain (SP) were enrolled into the treatment (TRT) stage and randomized to daily loratadine 10 mg or placebo for 7 days. SP was defined by Brief Pain Inventory as back or leg pain score ≥5 and a 2-point increase after pegfilgrastim. The primary end point of TRT was reduction of worst back or leg bone pain with loratadine, defined as a 2-point decrease after treatment compared to OBS. RESULTS: Two hundred thirteen patients were included in the final analysis. Incidence of SP was 30.5 %. The SP subset had a worse overall Functional Assessment of Cancer Therapy-Bone Pain score (33.9 vs. 51.7, p < 0.001) and a higher mean white blood cell count (15.4 vs. 8.4 K/cm(3), p = 0.013) following pegfilgrastim than those without SP. Forty-six patients were randomized in the TRT. Benefit was 77.3 % with loratadine and 62.5 % with placebo (p = 0.35). Baseline NSAID use was documented in four patients (18.2 %) in loratadine arm and two patients (8.3 %) in placebo arm, with baseline non-NSAID use documented in five (22.7 %) and six (25 %) patients, respectively. Eight additional patients used NSAIDS by day 8 compared to day 1 (six in the loratadine and two in the placebo arm). A total of six additional patients used non-NSAIDS by day 8 compared to day 1 (four in the loratadine and two in the placebo arm). CONCLUSIONS: Administration of prophylactic loratadine does not decrease the incidence of severe bone pain or improve quality of life in a high-risk patient population. ClinicalTrials.gov identifier: NCT01311336.

Tumor oncogene (KRAS) status and risk of venous thrombosis in patients with metastatic colorectal cancer.

BACKGROUND: Patients with metastatic colon cancer (mCRC) are at increased risk of venous thromboembolism (VTE). Limited preclinical data suggest that the oncogene (KRAS) mutational status of the tumor represents a plausible clinical link to systemic hypercoagulability in cancer patients. OBJECTIVES: To determine if a tumor genetic characteristic, KRAS mutational status, is associated with an increased risk of VTE in patients with mCRC. PATIENTS/METHODS: A retrospective cohort study of patients with mCRC and KRAS test results was conducted at multiple practice sites across New England in the United States. The primary outcome was a VTE event, defined as deep venous thrombosis (DVT) and/or pulmonary embolism (PE), either 6 months before or at any time after the diagnosis of mCRC. KRAS status (mutated vs. wild type) and other relevant predictors of thrombosis were collected. RESULTS: Of 172 histologically confirmed patients with mCRC, 40 developed a VTE (23.3%). Sixty-five patients (37.8%) had a mutant KRAS status. The incidence of VTE and DVT among patients with mutated KRAS was 32.3 and 23.1%, respectively. The corresponding incidence among patients with wild-type KRAS was 17.8 and 9.4%. Odd ratios for the association were 2.21 (95% CI, 1.08-4.53) for VTE and 2.62 (95% CI, 1.12-6.12) for DVT, and remained significant despite adjustment for Khorana score and bevacizumab use. CONCLUSION: Tumor mutant KRAS status is associated with an increased risk of VTE in patients with mCRC. The tumor genetic profile may represent a novel and important risk factor for thrombosis in patients with cancer.

Identification of inhibitors of a bacterial sigma factor using a new high-throughput screening assay.

Gram-negative bacteria are formidable pathogens because their cell envelope presents an adaptable barrier to environmental and host-mediated challenges. The stress response pathway controlled by the alternative sigma factor σ(E) is critical for maintenance of the cell envelope. Because σ(E) is required for the virulence or viability of several Gram-negative pathogens, it might be a useful target for antibiotic development. To determine if small molecules can inhibit the σ(E) pathway, and to permit high-throughput screening for antibiotic lead compounds, a σ(E) activity assay that is compatible with high-throughput screening was developed and validated. The screen employs a biological assay with positive readout. An Escherichia coli strain was engineered to express yellow fluorescent protein (YFP) under negative regulation by the σ(E) pathway, such that inhibitors of the pathway increase the production of YFP. To validate the screen, the reporter strain was used to identify σ(E) pathway inhibitors from a library of cyclic peptides. Biochemical characterization of one of the inhibitory cyclic peptides showed that it binds σ(E), inhibits RNA polymerase holoenzyme formation, and inhibits σ(E)-dependent transcription in vitro. These results demonstrate that alternative sigma factors can be inhibited by small molecules and enable high-throughput screening for inhibitors of the σ(E) pathway.

Statin therapy and levels of hemostatic factors in a healthy population: the Multi-Ethnic Study of Atherosclerosis.

BACKGROUND: HMG-CoA reductase inhibitors (statins) reduce the risk of venous thromboembolism (VTE) in healthy people. Statins reduce levels of inflammation biomarkers; however, the mechanism for the reduction in VTE risk is unknown. AIM: In a large cohort of healthy people, we studied associations of statin use with plasma hemostatic factors related to VTE risk. METHODS: Cross-sectional analyses were performed in the Multi-Ethnic Study of Atherosclerosis (MESA), a cohort study of 6814 healthy men and women aged 45-84 years, free of clinical cardiovascular disease at baseline; 1001 were using statins at baseline. Twenty-three warfarin users were excluded. Age, race and sex-adjusted mean hemostatic factor levels were compared between statin users and non-users, and multivariable linear regression models were used to assess associations of statin use with hemostatic factors, adjusted for age, race/ethnicity, education, income, aspirin use, hormone replacement therapy (in women), and major cardiovascular risk factors. RESULTS: Participants using statins had lower adjusted levels of D-dimer (- 9%), C-reactive protein (- 21%) and factor VIII (- 3%) than non-users (P < 0.05). Homocysteine and von Willebrand factor levels were non-significantly lower with statin use. Higher fibrinogen (2%) and plasminogen activator inhibitor-1 (22%) levels were observed among statin users than among non-users (P < 0.05). Further adjustment for LDL and triglyceride levels did not attenuate the observed differences in these factors with statin use. CONCLUSIONS: Findings of lower D-dimer, FVIII and C-reactive protein levels with statin use suggest hypotheses for mechanisms whereby statins might lower VTE risk. A prospective study or clinical trial linking these biochemical differences to VTE outcomes in statin users and non-users is warranted.

Perioperative DCF chemotherapy protocol for patients with gastroesophageal adenocarcinoma: correlation between response to treatment and outcome.

To determine whether metabolic or pathological response to preoperative chemotherapy can predict the relapse-free survival of gastroesophageal adenocarcinoma patients treated on a perioperative chemotherapy protocol. The prospectively collected data of a recently reported phase II trial of perioperative DCF chemotherapy (docetaxel/cisplatin/5-fluorouracil) were analyzed. Median relapse-free survival (RFS) was compared with the Wilcoxon rank-sum test between responders and non-responders according to defined metabolic (reduction in maximum standard uptake value of at least 35 %) and pathological (greater than 50 % tumor regression or ypN(0) status) criteria. A double-sided p value equal or inferior to 0.05 was considered significant. Patients were followed for a median of 807 days (95 % CI: 607-896). RFS was 576 days in metabolic non-responders versus not reached in metabolic responders (p 0.009) and 562 days in ypN+ versus not reached in ypN(0) patients (p 0.045). No statistically significant RFS difference was seen between low and high pathologic responders classified according to tumor regression criteria, although a trend was observed in favor of high pathologic responders. Simple metabolic and pathologic criteria used for the assessment of response to the preoperative part of perioperative chemotherapy can help to estimate the outcome of gastroesophageal adenocarcinoma patients.

Perioperative docetaxel, cisplatin, and 5-fluorouracil (DCF) for locally advanced esophageal and gastric adenocarcinoma: a multicenter phase II trial.

BACKGROUND: Although perioperative chemotherapy for esophagogastric adenocarcinoma (ADC) improves survival, the overall poor prognosis suggests that further refinement of treatment is required. Docetaxel, cisplatin, and 5-fluorouracil (5-FU) (DCF) is effective for metastatic ADC of the upper gastrointestinal (GI) tract; we thus sought to investigate the efficacy of this regimen in patients with resectable disease. PATIENTS AND METHODS: Patients with resectable ADC of the upper GI tract received DCF [docetaxel (Taxotere) 75 mg/m(2) I.V. day 1, cisplatin 75 mg/m(2) I.V. day 1, 5-FU 750 mg/m(2) continuous infusion for 120 h, every 3 weeks] for three cycles before and after resection. Primary end point was complete resection; secondary end points were response, toxicity, surgical morbidity, and overall survival. RESULTS: Forty-three patients with ADC of the esophagus (11), gastroesophageal junction (25), or stomach (7) started treatment and 86% completed all preoperative cycles with grade 3-4 toxicity arising in 47%. Metabolic response to chemotherapy (reduction in maximal standard uptake value >35%) was achieved in 25/33 (76%) patients. Surgery was carried out in 41/43 and complete resection was achieved in all 41 patients with pathologic complete response in 4/41. Postoperative chemotherapy was started in 29 patients and completed in 24. Three-year overall survival was 60%. CONCLUSION: Perioperative DCF is a tolerable and highly effective regimen for the treatment of esophagogastric ADC.

Lens changes in hereditary hyperferritinemia-cataract syndrome.

PURPOSE: To provide detailed description and illustration of the lens changes found in hereditary hyperferritinemia-cataract syndrome, a newly reported autosomal dominant condition. METHODS: Observational case reports. A 19-year-old man was referred for evaluation of possible hereditary hyperferritinemia-cataract syndrome. His serum ferritin level was increased at 1291 microg/L during a routine screening examination. Genetic analysis revealed mutation G51C on chromosome 19, predicting an altered iron response element in L-ferritin mRNA. Subsequent evaluation of his 46-year-old father revealed similar findings. RESULTS: Multiple breadcrumb-like nuclear and cortical lens opacities were seen in this father-son pair. These cases represent the first detailed description and illustration of hereditary hyperferritinemia-cataract syndrome cataracts in the ophthalmic literature. CONCLUSION: Hereditary hyperferritinemia-cataract syndrome can be associated with distinct breadcrumb-like lens opacities. Recognition of these characteristic cataracts may aid identification and study of patients with this unusual disorder and provide insight into mechanisms of cataract formation.

The Escherichia coli sigma(E)-dependent extracytoplasmic stress response is controlled by the regulated proteolysis of an anti-sigma factor.

The activity of the stress-responsive sigma factor, sigma(E), is induced by the extracytoplasmic accumulation of misfolded or unfolded protein. The inner membrane protein RseA is the central regulatory molecule in this signal transduction cascade and acts as a sigma(E)-specific anti-sigma factor. Here we show that sigma(E) activity is primarily determined by the ratio of RseA to sigma(E). RseA is rapidly degraded in response to extracytoplasmic stress, leading to an increase in the free pool of sigma(E) and initiation of the stress response. We present evidence that the putative inner membrane serine protease, DegS, is responsible for this regulated degradation of RseA.

Insect immunity. Isolation from the lepidopteran Heliothis virescens of a novel insect defensin with potent antifungal activity.

Lepidoptera have been reported to produce several antibacterial peptides in response to septic injury. However, in marked contrast to other insect groups, no inducible antifungal molecules had been described so far in this insect order. Surprisingly, also cysteine-rich antimicrobial peptides, which predominate in the antimicrobial defense of other insects, had not been discovered in Lepidoptera. Here we report the isolation from the hemolymph of immune induced larvae of the lepidopteran Heliothis virescens of a cysteine-rich molecule with exclusive antifungal activity. We have fully characterized this antifungal molecule, which has significant homology with the insect defensins, a large family of antibacterial peptides directed against Gram-positive strains. Interestingly, the novel peptide shows also similarities with the antifungal peptide drosomycin from Drosophila. Thus, Lepidoptera appear to have built their humoral immune response against bacteria on cecropins and attacins. In addition, we report that Lepidoptera have conferred antifungal properties to the well conserved structure of antibacterial insect defensins through amino acid replacements.

Engrailed (Gln50-->Lys) homeodomain-DNA complex at 1.9 A resolution: structural basis for enhanced affinity and altered specificity.

BACKGROUND: The homeodomain is one of the key DNA-binding motifs used in eukaryotic gene regulation, and homeodomain proteins play critical roles in development. The residue at position 50 of many homeodomains appears to determine the differential DNA-binding specificity, helping to distinguish among binding sites of the form TAATNN. However, the precise role(s) of residue 50 in the differential recognition of alternative sites has not been clear. None of the previously determined structures of homeodomain-DNA complexes has shown evidence for a stable hydrogen bond between residue 50 and a base, and there has been much discussion, based in part on NMR studies, about the potential importance of water-mediated contacts. This study was initiated to help clarify some of these issues. RESULTS: The crystal structure of a complex containing the engrailed Gln50-->Lys variant (QK50) with its optimal binding site TAATCC (versus TAATTA for the wild-type protein) has been determined at 1.9 A resolution. The overall structure of the QK50 variant is very similar to that of the wild-type complex, but the sidechain of Lys50 projects directly into the major groove and makes several hydrogen bonds to the O6 and N7 atoms of the guanines at base pairs 5 and 6. Lys50 also makes an additional water-mediated contact with the guanine at base pair 5 and has an alternative conformation that allows a hydrogen bond with the O4 of the thymine at base pair 4. CONCLUSIONS: The structural context provided by the folding and docking of the engrailed homeodomain allows Lys50 to make remarkably favorable contacts with the guanines at base pairs 5 and 6 of the binding site. Although many different residues occur at position 50 in different homeodomains, and although numerous position 50 variants have been constructed, the most striking examples of altered specificity usually involve introducing or removing a lysine sidechain from position 50. This high-resolution structure also confirms the critical role of Asn51 in homeodomain-DNA recognition and further clarifies the roles of water molecules near residues 50 and 51.

Specificity of minor-groove and major-groove interactions in a homeodomain-DNA complex.

To assess the importance of minor-groove and major-groove interactions in homeodomain-DNA recognition, the binding properties of variants of the altered-specificity engrailed homeodomain, containing Lys50, and its DNA site TAATCC were determined. This homeodomain contacts bases in the minor groove of the DNA using Arg3 and Arg5 from its N-terminal arm and contacts bases in the major groove of the DNA using Ile47, Lys50, and Asn51 from its third alpha-helix. Mutation of Arg3 or Ile47 to alanine reduces binding affinity 10-20-fold while mutation of Arg5, Asn51, or Lys50 to alanine reduces binding affinity > 100-fold, indicating that both minor-groove and major-groove interactions contribute to the overall binding energy. Binding site selections and affinity measurements show that the homeodomain can also discriminate among different base pairs in the minor groove and the major groove. However, the interactions between Lys50 of the recognition helix and the major-groove edges of base pairs 5 and 6 are more specific than interactions mediated by Arg3 and Arg5 in the N-terminal arm and the minor-groove edges of base pairs 1 and 2.

Differential DNA-binding specificity of the engrailed homeodomain: the role of residue 50.

To assess the importance of residue 50 in determining the binding specificity of the homeodomain from the engrailed transcription factor of Drosophila, the DNA-binding properties of isolated homeodomains containing glutamine (wild type), alanine, and lysine at this position have been studied. In binding site selection experiments using the wild-type engrailed homeodomain, TAATTA was identified as a high-affinity, consensus binding site. When the glutamine at position 50 was replaced by a lysine (QK50), the binding site preference changed to TAATCC. The half-life and affinity of the complex between the QK50 protein and a DNA site containing TAATCC were increased significantly compared to the half-life and affinity of the complex between the wild-type protein and a TAATTA site. This suggests that Lys50 forms a more favorable interaction with the TAATCC DNA than Gln50 does with the TAATTA site. In fact, the wild-type Gln50 side chain (which forms a hydrophobic interaction with the last A:T base pair of the TAATTA site in the cocrystal structure [Kissinger, C. R., Liu, B., Martin-Blanco, E., Kornberg, T. B., & Pabo, C. O. (1990) Cell 63, 579-590]) appears to play only a small role in determining binding affinity and specificity for the TAATTA site, as the QA50 mutant has only a 2-fold reduced affinity for the TAATTA site and discriminates between the TAATTA and TAATCC sites as well as the wild-type protein. As a result, determinants in addition to Gln50 must be involved in establishing the differential binding specificity of the engrailed homeodomain.

Sequence and domain structure of talin.

Talin is a high-molecular-weight cytoskeletal protein concentrated at regions of cell-substratum contact and, in lymphocytes, at cell-cell contacts. Integrin receptors are involved in the attachment of adherent cells to extracellular matrices and of lymphocytes to other cells. In these situations, talin codistributes with concentrations of integrins in the cell surface membrane. Furthermore, in vitro binding studies suggest that integrins bind to talin, although with low affinity. Talin also binds with high affinity to vinculin, another cytoskeletal protein concentrated at points of cell adhesion. Finally, talin is a substrate for the Ca2(+)-activated protease, calpain II, which is also concentrated at points of cell-substratum contact. To learn more about the structure of talin and its involvement in transmembrane connections between extracellular adhesions and the cytoskeleton, we have cloned and sequenced murine talin. We describe a model for the structure of talin based on this sequence and other data. Homologies between talin and other proteins define a novel family of submembranous cytoskeleton-associated proteins all apparently involved in connections to the plasma membrane.