Development of a Selective Peptide Macrocycle Inhibitor of Coagulation Factor XII toward the Generation of a Safe Antithrombotic Therapy.

Inhibition of coagulation factor XII (FXII) activity represents an attractive approach for the treatment and prevention of thrombotic diseases. The few existing FXII inhibitors suffer from low selectivity. Using phage display combined to rational design, we developed a potent inhibitor of FXII with more than 100-fold selectivity over related proteases. The highly selective peptide macrocycle is a promising candidate for the control of FXII activity in antithrombotic therapy and a valuable tool in hematology research.

Impact of antiretroviral therapy (ART), immunosuppression and viraemia on lipid levels: the D:A:D study

Purpose of the study: To investigate the impact of ART, HIV viremia and immunosuppression on triglyceride (TG), total cholesterol (TC) and high density lipoprotein cholesterol (HDL-C) levels. Methods: We considered the cross-sectional associations between TG, TC and HDL-C (mmol/l; first available measurement on/after enrolment in the D:A:D study) and use of ART, HIV viral load (VL; copies/ml), and CD4 count (cells/mm3) measured at the same time. TG was log10 transformed to ensure normality. Analyses were performed using linear regression and adjusted for other factors known to impact lipid levels (table footnote). ART and VL status were combined (off ART&VL _100,000, off ART&VL B100,000, on ART&VL B500, on ART&VL _500), current and nadir CD4 count were categorised as B200, 200_349, 350_499 and _500. Summary of results: 44,322/49,734 participants in the D:A:D Study (89.1%) contributed a TG measurement (median; IQR 1.52; 1.00_ 2.45), 45,169 (90.8%) a TC measurement (4.80; 4.00_5.70) and 38,604 (77.6%) a HDL-C measurement (1.12; 0.90_1.40). Most participants were male (74%), of white ethnicity (51%), without AIDS (78%), were not receiving lipid-lowering drugs (4%) and were ART experienced (61%) with 47% previously exposed to PIs, 61% previously exposed to NRTIs and 29% previously exposed to NNRTIs. The median (IQR) age, current CD4 count and CD4 nadir were 38 (36_45) years, 400 (242_590) cells/ml and 240 (100_410) cells/ml respectively. Compared to those on ART with a suppressed VL, all lipids were lower for those off ART (Table); non-suppressive ART was also associated with lower TC and HDL-C levels (no impact on TG). A low current CD4 count was associated with lower lipid levels, whereas a low nadir CD4 count was associated with higher TC and TG levels. Prior AIDS diagnosis was associated with higher TG and TC, but lower HDL-C levels. Conclusion: Although specific drug classes were not considered, lipid levels are considerably higher in those on a suppressive ART regimen. The higher TC/TG and lower HDL-C levels seen among those with low nadir CD4 count and with a prior AIDS diagnosis suggests severe immunosuppression may be associated with dyslipidaemia over the long-term.

MGMT promoter methylation is prognostic but not predictive for outcome to adjuvant PCV chemotherapy in anaplastic oligodendroglial tumors: a report from EORTC Brain Tumor Group Study 26951.

PURPOSE: O6-methylguanine-methyltransferase (MGMT) promoter methylation has been shown to predict survival of patients with glioblastomas if temozolomide is added to radiotherapy (RT). It is unknown if MGMT promoter methylation is also predictive to outcome to RT followed by adjuvant procarbazine, lomustine, and vincristine (PCV) chemotherapy in patients with anaplastic oligodendroglial tumors (AOT). PATIENTS AND METHODS: In the European Organisation for the Research and Treatment of Cancer study 26951, 368 patients with AOT were randomly assigned to either RT alone or to RT followed by adjuvant PCV. From 165 patients of this study, formalin-fixed, paraffin-embedded tumor tissue was available for MGMT promoter methylation analysis. This was investigated with methylation specific multiplex ligation-dependent probe amplification. RESULTS: In 152 cases, an MGMT result was obtained, in 121 (80%) cases MGMT promoter methylation was observed. Methylation strongly correlated with combined loss of chromosome 1p and 19q loss (P = .00043). In multivariate analysis, MGMT promoter methylation, 1p/19q codeletion, tumor necrosis, and extent of resection were independent prognostic factors. The prognostic significance of MGMT promoter methylation was equally strong in the RT arm and the RT/PCV arm for both progression-free survival and overall survival. In tumors diagnosed at central pathology review as glioblastoma, no prognostic effect of MGMT promoter methylation was observed. CONCLUSION: In this study, on patients with AOT MGMT promoter methylation was of prognostic significance and did not have predictive significance for outcome to adjuvant PCV chemotherapy. The biologic effect of MGMT promoter methylation or pathogenetic features associated with MGMT promoter methylation may be different for AOT compared with glioblastoma.

Use of a combined ex vivo/in vivo population approach for screening of human genes involved in the human immunodeficiency virus type 1 life cycle for variants influencing disease progression.

Humans differ substantially with respect to susceptibility to human immunodeficiency virus type 1 (HIV-1). We evaluated variants of nine host genes participating in the viral life cycle for their role in modulating HIV-1 infection. Alleles were assessed ex vivo for their impact on viral replication in purified CD4 T cells from healthy blood donors (n = 128). Thereafter, candidate alleles were assessed in vivo in a cohort of HIV-1-infected individuals (n = 851) not receiving potent antiretroviral therapy. As a benchmark test, we tested 12 previously reported host genetic variants influencing HIV-1 infection as well as single nucleotide polymorphisms in the nine candidate genes. This led to the proposition of three alleles of PML, TSG101, and PPIA as potentially associated with differences in progression of HIV-1 disease. In a model considering the combined effects of new and previously reported gene variants, we estimated that their effect might be responsible for lengthening or shortening by up to 2.8 years the period from 500 CD4 T cells/mul to <200 CD4 T cells/mul.

Effects of a treatment gap during adjuvant chemotherapy in node-positive breast cancer: results of International Breast Cancer Study Group (IBCSG) Trials 13-93 and 14-93.

BACKGROUND: The International Breast Cancer Study Group (IBCSG) conducted two complementary randomized trials to assess whether a treatment-free gap during adjuvant chemotherapy influenced outcome. PATIENTS AND METHODS: From 1993 to 1999, IBCSG Trials 13-93 and 14-93 enrolled 2215 premenopausal and postmenopausal women with axillary node-positive, operable breast cancer. All patients received cyclophosphamide (Cytoxan, C) plus either doxorubicin (Adriamycin, A) or epirubicin (E) for four courses followed immediately (No Gap) or after a 16-week delay (Gap) by classical cyclophosphamide, methotrexate, and fluorouracil (CMF) for three courses. The median follow-up was 7.7 years. RESULTS: The Gap and No-Gap groups had similar disease-free survival (DFS) and overall survival (OS). No identified subgroup showed a statistically significant difference, but exploratory subgroup analysis noted a trend towards decreased DFS for Gap compared with No Gap for women with estrogen receptor (ER)-negative tumors not receiving tamoxifen, especially evident during the first 2 years. CONCLUSIONS: A 16-week gap between adjuvant AC/EC and CMF provided no benefit and may have increased early recurrence rates in patients with ER-negative tumors.

Major bleeding risk in anticoagulated patients receiving concomitant antiplatelet therapy: A prospective study.

INTRODUCTION: Current literature suggesting a higher bleeding risk during combination therapy compared to oral anticoagulation alone is primarily based on retrospective studies or specific populations. We aimed to prospectively evaluate whether unselected medical patients on oral anticoagulation have an increased risk of bleeding when on concomitant antiplatelet therapy. MATERIAL AND METHODS: We prospectively studied consecutive adult medical patients who were discharged on oral anticoagulants between 01/2008 and 03/2009 from a Swiss university hospital. The primary outcome was the time to a first major bleed on oral anticoagulation within 12months, adjusted for age, international normalized ratio target, number of medications, and history of myocardial infarction and major bleeding. RESULTS: Among the 515 included anticoagulated patients, the incidence rate of a first major bleed was 8.2 per 100 patient-years. Overall, 161 patients (31.3%) were on both anticoagulant and antiplatelet therapy, and these patients had a similar incidence rate of major bleeding compared to patients on oral anticoagulation alone (7.6 vs. 8.4 per 100 patient-years, P=0.81). In a multivariate analysis, the association of concomitant antiplatelet therapy with the risk of major bleeding was not statistically significant (hazard ratio 0.89, 95% confidence interval, 0.37-2.10). CONCLUSIONS: The risk of bleeding in patients receiving oral anticoagulants combined with antiplatelet therapy was similar to patients receiving oral anticoagulants alone, suggesting that the incremental bleeding risk of combination therapy might not be clinically significant.

Phase 3 randomized trial on larynx preservation comparing sequential vs alternating chemotherapy and radiotherapy.

BACKGROUND: Both induction chemotherapy followed by irradiation and concurrent chemotherapy and radiotherapy have been reported as valuable alternatives to total laryngectomy in patients with advanced larynx or hypopharynx cancer. We report results of the randomized phase 3 trial 24954 from the European Organization for Research and Treatment of Cancer. METHODS: Patients with resectable advanced squamous cell carcinoma of the larynx (tumor stage T3-T4) or hypopharynx (T2-T4), with regional lymph nodes in the neck staged as N0-N2 and with no metastasis, were randomly assigned to treatment in the sequential (or control) or the alternating (or experimental) arm. In the sequential arm, patients with a 50% or more reduction in primary tumor size after two cycles of cisplatin and 5-fluorouracil received another two cycles, followed by radiotherapy (70 Gy total). In the alternating arm, a total of four cycles of cisplatin and 5-fluorouracil (in weeks 1, 4, 7, and 10) were alternated with radiotherapy with 20 Gy during the three 2-week intervals between chemotherapy cycles (60 Gy total). All nonresponders underwent salvage surgery and postoperative radiotherapy. The Kaplan-Meier method was used to obtain time-to-event data. RESULTS: The 450 patients were randomly assigned to treatment (224 to the sequential arm and 226 to the alternating arm). Median follow-up was 6.5 years. Survival with a functional larynx was similar in sequential and alternating arms (hazard ratio of death and/or event = 0.85, 95% confidence interval = 0.68 to 1.06), as were median overall survival (4.4 and 5.1 years, respectively) and median progression-free interval (3.0 and 3.1 years, respectively). Grade 3 or 4 mucositis occurred in 64 (32%) of the 200 patients in the sequential arm who received radiotherapy and in 47 (21%) of the 220 patients in the alternating arm. Late severe edema and/or fibrosis was observed in 32 (16%) patients in the sequential arm and in 25 (11%) in the alternating arm. CONCLUSIONS: Larynx preservation, progression-free interval, and overall survival were similar in both arms, as were acute and late toxic effects.

Predictive value of the MGMT promoter methylation status in metastatic melanoma patients receiving first-line temozolomide plus bevacizumab in the trial SAKK 50/07.

The O6-methylguanine-DNA-methyltransferase (MGMT) promoter methylation status is a predictive parameter for the response of malignant gliomas to alkylating agents such as temozolomide. First clinical trials with temozolomide plus bevacizumab therapy in metastatic melanoma patients are ongoing, although the predictive value of the MGMT promoter methylation status in this setting remains unclear. We assessed MGMT promoter methylation in formalin-fixed, primary tumor tissue of metastatic melanoma patients treated with first-line temozolomide and bevacizumab from the trial SAKK 50/07 by methylation-specific polymerase chain reaction. In addition, the MGMT expression levels were also analyzed by MGMT immunohistochemistry. Eleven of 42 primary melanomas (26%) revealed a methylated MGMT promoter. Promoter methylation was significantly associated with response rates CR + PR versus SD + PD according to RECIST (response evaluation criteria in solid tumors) (p<0.05) with a trend to prolonged median progression-free survival (8.1 versus 3.4 months, p>0.05). Immunohistochemically different protein expression patterns with heterogeneous and homogeneous nuclear MGMT expression were identified. Negative MGMT expression levels were associated with overall disease stabilization CR+PR+SD versus PD (p=0.05). There was only a poor correlation between MGMT methylation and lack of MGMT expression. A significant proportion of melanomas have a methylated MGMT promoter. The MGMT promoter methylation status may be a promising predictive marker for temozolomide therapy in metastatic melanoma patients. Larger sample sizes may help to validate significant differences in survival type endpoints.

Topical therapy is underused in patients with ulcerative colitis.

The availability of new topical preparations for the treatment of left sided ulcerative colitis offers a therapy optimization for many patients. Rectal application of steroids and 5-aminosalicylic acid (5-ASA) is associated with fewer side effects and has a higher therapeutic efficacy in left-sided colitis as compared to a systemic therapy. Therefore, we were interested in the use of topical therapy in patients with ulcerative colitis. The key question was whether topical treatment is more frequently used than oral therapy in patients with proctitis and left sided colitis. Data of 800 patients of the Swiss IBD cohort study were analyzed. Sixteen percent of patients of the cohort had proctitis, 21% proctosigmoiditis and 41% pancolitis. Topical therapy with 5-ASA or corticosteroids was given in 26% of patients with proctitis, a combined systemic and topical treatment was given in 13%, whereas systemic treatment with 5-ASA without topical treatment was given in 29%. Proportion of topical drug use decreased with respect to disease extension from 39% for proctitis to 13.1% for pancolitis (P=0.001). Patients with severe colitis received a significantly higher dose of topical 5-ASA than patients in remission. Side effects of topical or systemic 5-ASA or budesonide treatment were less frequently seen compared to other medications. Topical treatment was frequently stopped over time. The quality of life was the same in patients with limited disease compared to patients with pancolitis. Topical treatment in proctitis patients was underused in Switzerland. Since topical treatment is safe and effective it should be used to a larger extend.

In vivo imaging of T cell delivery to tumors after adoptive transfer therapy.

Adoptive transfer therapy of in vitro-expanded tumor-specific cytolytic T lymphocytes (CTLs) can mediate objective cancer regression in patients. Yet, technical limitations hamper precise monitoring of posttherapy T cell responses. Here we show in a mouse model that fused single photon emission computed tomography and x-ray computed tomography allows quantitative whole-body imaging of (111)In-oxine-labeled CTLs at tumor sites. Assessment of CTL localization is rapid, noninvasive, three-dimensional, and can be repeated for longitudinal analyses. We compared the effects of lymphodepletion before adoptive transfer on CTL recruitment and report that combined treatment increased intratumoral delivery of CTLs and improved antitumor efficacy. Because (111)In-oxine is a Food and Drug Administration-approved clinical agent, and human SPECT-CT systems are available, this approach should be clinically translatable, insofar as it may assess the efficacy of immunization procedures in individual patients and lead to development of more effective therapies.

Association of the hemochromatosis gene with pazopanib-induced transaminase elevation in renal cell carcinoma.

BACKGROUND & AIMS: Pazopanib has demonstrated clinical benefit in patients with advanced renal cell carcinoma (RCC) and is generally well tolerated. However, transaminase elevations have commonly been observed. This 2-stage study sought to identify genetic determinants of alanine transaminase (ALT) elevations in pazopanib-treated white patients with RCC.¦METHODS: Data from two separate clinical studies were used to examine the association of genetic polymorphisms with maximum on-treatment ALT levels.¦RESULTS: Of 6852 polymorphisms in 282 candidate genes examined in an exploratory dataset of 115 patients, 92 polymorphisms in 40 genes were significantly associated with ALT elevation (p<0.01). Two markers (rs2858996 and rs707889) in the HFE gene, which are not yet known to be associated with hemochromatosis, showed evidence for replication. Because of multiple comparisons, there was a 12% likelihood the replication occurred by chance. These two markers demonstrated strong linkage disequilibrium (r(2)=0.99). In the combined dataset, median (25-75th percentile) maximum ALT values were 1.2 (0.7-1.9), 1.1 (0.8-2.5), and 5.4 (1.9-7.6)×ULN for rs2858996 GG (n=148), GT (n=82), and TT (n=1 2) genotypes, respectively. All 12 TT patients had a maximum ALT>ULN, and 8 (67%) had ALT≥3×ULN. The odds ratio (95% CI) for ALT≥3×ULN for TT genotype was 39.7 (2.2-703.7) compared with other genotypes. As a predictor of ALT≥3×ULN, the TT genotype had a negative predictive value of 0.83 and positive predictive value of 0.67. No TT patients developed liver failure.¦CONCLUSIONS: The rs2858996/rs707889 polymorphisms in the HFE gene may be associated with reversible ALT elevation in pazo-panib-treated patients with RCC.

p27 and Skp2 immunoreactivity and its clinical significance with endocrine and chemo-endocrine treatments in node-negative early breast cancer.

BACKGROUND: Low p27 and high Skp2 immunoreactivity are associated with a poor prognosis and other poor prognostic features including resistant phenotypes and antiestrogen drug resistance. We investigated these proteins in two International Breast Cancer Study Group trials studying node-negative early breast cancer. PATIENTS AND METHODS: Trial VIII compared chemotherapy followed by goserelin with either modality alone in premenopausal patients. Trial IX compared chemotherapy followed by tamoxifen with tamoxifen alone in postmenopausal patients. Central Pathology Office assessed p27 and Skp2 expression in the primary tumor by immunohistochemistry among 1631 (60%) trial patients. RESULTS: p27 and Skp2 were inversely related; 13% of tumors expressed low p27 and high Skp2. Low p27 and high Skp2 were associated with unfavorable prognostic factors including larger size and higher grade tumors, absence of estrogen receptor and progesterone receptor, human epidermal growth factor receptor 2 overexpression and high Ki-67 (each P < 0.05). Low p27 and high Skp2 were not associated with disease-free survival (P = 0.42 and P = 0.48, respectively). The relative effects of chemo-endocrine versus endocrine therapy were similar regardless of p27 or Skp2. CONCLUSIONS: We confirm the association of low p27 and high Skp2 with other poor prognostic features, but found no predictive or prognostic value, and therefore do not recommend routine determination of p27 and Skp2 for node-negative breast cancer.

Clinical, molecular, and prognostic significance of WHO type inv(3)(q21q26.2)/t(3;3)(q21;q26.2) and various other 3q abnormalities in acute myeloid leukemia.

PURPOSE: Acute myeloid leukemia (AML) with inv(3)(q21q26.2)/t(3;3)(q21;q26.2) [inv(3)/t(3;3)] is recognized as a distinctive entity in the WHO classification. Risk assignment and clinical and genetic characterization of AML with chromosome 3q abnormalities other than inv(3)/t(3;3) remain largely unresolved. PATIENTS AND METHODS: Cytogenetics, molecular genetics, therapy response, and outcome analysis were performed in 6,515 newly diagnosed adult AML patients. Patients were treated on Dutch-Belgian Hemato-Oncology Cooperative Group/Swiss Group for Clinical Cancer Research (HOVON/SAKK; n = 3,501) and German-Austrian Acute Myeloid Leukemia Study Group (AMLSG; n = 3,014) protocols. EVI1 and MDS1/EVI1 expression was determined by real-time quantitative polymerase chain reaction. RESULTS: 3q abnormalities were detected in 4.4% of AML patients (288 of 6,515). Four distinct groups were defined: A: inv(3)/t(3;3), 32%; B: balanced t(3q26), 18%; C: balanced t(3q21), 7%; and D: other 3q abnormalities, 43%. Monosomy 7 was the most common additional aberration in groups (A), 66%; (B), 31%; and (D), 37%. N-RAS mutations and dissociate EVI1 versus MDS1/EVI1 overexpression were associated with inv(3)/t(3;3). Patients with inv(3)/t(3;3) and balanced t(3q21) at diagnosis presented with higher WBC and platelet counts. In multivariable analysis, only inv(3)/t(3;3), but not t(3q26) and t(3q21), predicted reduced relapse-free survival (hazard ratio [HR], 1.99; P < .001) and overall survival (HR, 1.4; P = .006). This adverse prognostic impact of inv(3)/t(3;3) was enhanced by additional monosomy 7. Group D 3q aberrant AML also had a poor outcome related to the coexistence of complex and/or monosomal karyotypes and cryptic inv(3)/t(3;3). CONCLUSION: Various categories of 3q abnormalities in AML can be distinguished according to their clinical, hematologic, and genetic features. AML with inv(3)/t(3;3) represents a distinctive subgroup with unfavorable prognosis.

Topical therapy is underused in patients with ulcerative colitis.

The availability of new topical preparations for the treatment of left sided ulcerative colitis offers a therapy optimization for many patients. Rectal application of steroids and 5-aminosalicylic acid (5-ASA) is associated with fewer side effects and has a higher therapeutic efficacy in left-sided colitis as compared to a systemic therapy. Therefore, we were interested in the use of topical therapy in patients with ulcerative colitis. The key question was whether topical treatment is more frequently used than oral therapy in patients with proctitis and left sided colitis. Data of 800 patients of the Swiss IBD cohort study were analyzed. Sixteen percent of patients of the cohort had proctitis, 21% proctosigmoiditis and 41% pancolitis. Topical therapy with 5-ASA or corticosteroids was given in 26% of patients with proctitis, a combined systemic and topical treatment was given in 13%, whereas systemic treatment with 5-ASA without topical treatment was given in 29%. Proportion of topical drug use decreased with respect to disease extension from 39% for proctitis to 13.1% for pancolitis (P=0.001). Patients with severe colitis received a significantly higher dose of topical 5-ASA than patients in remission. Side effects of topical or systemic 5-ASA or budesonide treatment were less frequently seen compared to other medications. Topical treatment was frequently stopped over time. The quality of life was the same in patients with limited disease compared to patients with pancolitis. Topical treatment in proctitis patients was underused in Switzerland. Since topical treatment is safe and effective it should be used to a larger extend.

Is adjuvant chemotherapy of benefit for postmenopausal women who receive endocrine treatment for highly endocrine-responsive, node-positive breast cancer? International Breast Cancer Study Group Trials VII and 12-93.

To compare the efficacy of chemoendocrine treatment with that of endocrine treatment (ET) alone for postmenopausal women with highly endocrine responsive breast cancer. In the International Breast Cancer Study Group (IBCSG) Trials VII and 12-93, postmenopausal women with node-positive, estrogen receptor (ER)-positive or ER-negative, operable breast cancer were randomized to receive either chemotherapy or endocrine therapy or combined chemoendocrine treatment. Results were analyzed overall in the cohort of 893 patients with endocrine-responsive disease, and according to prospectively defined categories of ER, age and nodal status. STEPP analyses assessed chemotherapy effect. The median follow-up was 13 years. Adding chemotherapy reduced the relative risk of a disease-free survival event by 19% (P = 0.02) compared with ET alone. STEPP analyses showed little effect of chemotherapy for tumors with high levels of ER expression (P = 0.07), or for the cohort with one positive node (P = 0.03). Chemotherapy significantly improves disease-free survival for postmenopausal women with endocrine-responsive breast cancer, but the magnitude of the effect is substantially attenuated if ER levels are high.