Expression of MYC, IgM, As Well As Non-Germinal Centre B-Cell Like Immunophenotype and Positive Immunofish Index Predict a Worse Progression Free Survival and Overall Survival in a Series of 670 De Novo Diffuse Large B-Cell Lymphomas Included in Clinical Trials: A GELA Study of the 2003 Program

Introduction: Diffuse large B-cell lymphomas (DLBCL) represent a heterogeneous disease with variable clinical outcome. Identifying phenotypic biomarkers of tumor cells on paraffin sections that predict different clinical outcome remain an important goal that may also help to better understand the biology of this lymphoma. Differentiating non-germinal centre B-cell-like (non-GCB) from Germinal Centre B-cell-like (GCB) DLBCL according to Hans algorithm has been considered as an important immunohistochemical biomarker with prognostic value among patients treated with R-CHOP although not reproducibly found by all groups. Gene expression studies have also shown that IgM expression might be used as a surrogate for the GCB and ABC subtypes with a strong preferential expression of IgM in ABC DLBCL subtype. ImmunoFISH index based on the differential expression of MUM-1, FOXP1 by immunohistochemistry and on the BCL6 rearrangement by FISH has been previously reported (C Copie-Bergman, J Clin Oncol. 2009;27:5573-9) as prognostic in an homogeneous series of DLBCL treated with R-CHOP. In addition, oncogenic MYC protein overexpression by immunohistochemistry may represent an easy tool to identify the consequences of MYC deregulation in DLBCL. Our aim was to analyse by immunohistochemistry the prognostic relevance of MYC, IgM, GCB/nonGCB subtype and ImmunoFISH index in a large series of de novo DLBCL treated with Rituximab (R)-chemotherapy (anthracyclin based) included in the 2003 program of the Groupe d'Etude des Lymphomes de l'Adulte (GELA) trials. Methods: The 2003 program included patients with de novo CD20+ DLBCL enrolled in 6 different LNH-03 GELA trials (LNH-03-1B, -B, -3B, 39B, -6B, 7B) stratifying patients according to age and age-adjusted IPI. Tumor samples were analyzed by immunohistochemistry using CD10, BCL6, MUM1, FOXP1 (according to Barrans threshold), MYC, IgM antibodies on tissue microarrays and by FISH using BCL6 split signal DNA probes. Considering evaluable Hans score, 670 patients were included in the study with 237 (35.4%) receiving intensive R-ACVBP regimen and 433 (64.6%) R-CHOP/R-mini-CHOP. Results: 304 (45.4%) DLBCL were classified as GCB and 366 (54.6%) as non-GCB according to Hans algorithm. 337/567 cases (59.4%) were positive for the ImmunoFISH index (i.e. two out of the three markers positive: MUM1 protein positive, FOXP1 protein Variable or Strong, BCL6 rearrangement). Immunofish index was preferentially positive in the non-GCB subtype (81.3%) compared to the GCB subtype (31.2%), (p<0.001). IgM was recorded as positive in tumor cells in 351/637 (52.4%) DLBCL cases with a preferential expression in non-GCB 195 (53.3%) vs GCB subtype 100(32.9%), p<0.001). MYC was positive in 170/577 (29.5%) cases with a 40% cut-off and in 44/577 (14.2%) cases with a cut-off of 70%. There was no preferential expression of MYC among GCB or non-GCB subtype (p>0.4) for both cut-offs. Progression-free Survival (PFS) was significantly worse among patients with high IPI score (p<0.0001), IgM positive tumor (p<0.0001), MYC positive tumor with a 40% threshold (p<0.001), ImmunoFISH positive index (p<0.002), non-GCB DLBCL subtype (p<0.0001). Overall Survival (OS) was also significantly worse among patients with high IPI score (p<0.0001), IgM positive tumor (p=0.02), MYC positive tumor with a 40% threshold (p<0.01), ImmunoFISH positive index (p=0.02), non-GCB DLBCL subtype (p<0.0001). All significant parameters were included in a multivariate analysis using Cox Model and in addition to IPI, only the GCB/non-GCB subtype according to Hans algorithm predicted significantly a worse PFS among non-GCB subgroup (HR 1.9 [1.3-2.8] p=0.002) as well as a worse OS (HR 2.0 [1.3-3.2], p=0.003). This strong prognostic value of non-GCB subtyping was confirmed considering only patients treated with R- CHOP for PFS (HR 2.1 [1.4-3.3], p=0.001) and for OS (HR 2.3 [1.3-3.8], p=0.002). Conclusion: Our study on a large series of patients included in trials confirmed the relevance of immunohistochemistry as a useful tool to identify significant prognostic biomarkers for clinical use. We show here that IgM and MYC might be useful prognostic biomarkers. In addition, we confirmed in this series the prognostic value of the ImmunoFISH index. Above all, we fully validated the strong and independent prognostic value of the Hans algorithm, daily used by the pathologists to subtype DLBCL.

Outcome of smoking cessation counselling of HIV-positive persons by HIV care physicians.

OBJECTIVES: Smoking is the most prevalent modifiable risk factor for cardiovascular diseases among HIV-positive persons. We assessed the effect on smoking cessation of training HIV care physicians in counselling. METHODS: The Swiss HIV Cohort Study (SHCS) is a multicentre prospective observational database. Our single-centre intervention at the Zurich centre included a half day of standardized training for physicians in counselling and in the pharmacotherapy of smokers, and a physicians' checklist for semi-annual documentation of their counselling. Smoking status was then compared between participants at the Zurich centre and other institutions. We used marginal logistic regression models with exchangeable correlation structure and robust standard errors to estimate the odds of smoking cessation and relapse. RESULTS: Between April 2000 and December 2010, 11 056 SHCS participants had 121 238 semi-annual visits and 64 118 person-years of follow-up. The prevalence of smoking decreased from 60 to 43%. During the intervention at the Zurich centre from November 2007 to December 2009, 1689 participants in this centre had 6068 cohort visits. These participants were more likely to stop smoking [odds ratio (OR) 1.23; 95% confidence interval (CI) 1.07-1.42; P=0.004] and had fewer relapses (OR 0.75; 95% CI 0.61-0.92; P=0.007) than participants at other SHCS institutions. The effect of the intervention was stronger than the calendar time effect (OR 1.19 vs. 1.04 per year, respectively). Middle-aged participants, injecting drug users, and participants with psychiatric problems or with higher alcohol consumption were less likely to stop smoking, whereas persons with a prior cardiovascular event were more likely to stop smoking. CONCLUSIONS: An institution-wide training programme for HIV care physicians in smoking cessation counselling led to increased smoking cessation and fewer relapses.

Phase I trial combining temozolomide plus lapatinib for the treatment of brain metastases in patients with HER2-positive metastatic breast cancer: the LAPTEM trial.

BACKGROUND: Brain metastases (BMs) pose a clinical challenge in breast cancer (BC). Lapatinib or temozolomide showed activity in BM. Our study assessed the combination of both drugs as treatment for patients with HER2-positive BC and BM. METHODS: Eighteen patients were enrolled, with sixteen of them having recurrent or progressive BM. Any type of previous therapy was allowed, and disease was assessed by gadolinium (Gd)-enhanced magnetic resonance imaging (MRI). The primary end points were the evaluation of the dose-limiting toxicities (DLTs) and the determination of the maximum-tolerated dose (MTD). The secondary end points included objective response rate, clinical benefit and duration of response. RESULTS: The lapatinib-temozolomide regimen showed a favorable toxicity profile because the MTD could not be reached. The most common adverse events (AEs) were fatigue, diarrhea and constipation. Disease stabilization was achieved in 10 out of 15 assessable patients. The estimated median survival time for the 16 patients with BM reached 10.94 months (95% CI: 1.09-20.79), whereas the median progression-free survival time was 2.60 months [95% confidence interval (CI): 1.82-3.37]. CONCLUSIONS: The lapatinib-temozolomide combination is well tolerated. Preliminary evidence of clinical activity was observed in a heavily pretreated population, as indicated by the volumetric reductions occurring in brain lesions.

Design, conduct, and analyses of Breast International Group (BIG) 1-98: a randomized, double-blind, phase-III study comparing letrozole and tamoxifen as adjuvant endocrine therapy for postmenopausal women with receptor-positive, early breast cancer.

BACKGROUND: Aromatase inhibitors provide superior disease control when compared with tamoxifen as adjuvant therapy for postmenopausal women with endocrine-responsive early breast cancer. PURPOSE: To present the design, history, and analytic challenges of the Breast International Group (BIG) 1-98 trial: an international, multicenter, randomized, double-blind, phase-III study comparing the aromatase inhibitor letrozole with tamoxifen in this clinical setting. METHODS: From 1998-2003, BIG 1-98 enrolled 8028 women to receive monotherapy with either tamoxifen or letrozole for 5 years, or sequential therapy of 2 years of one agent followed by 3 years of the other. Randomization to one of four treatment groups permitted two complementary analyses to be conducted several years apart. The first, reported in 2005, provided a head-to-head comparison of letrozole versus tamoxifen. Statistical power was increased by an enriched design, which included patients who were assigned sequential treatments until the time of the treatment switch. The second, reported in late 2008, used a conditional landmark approach to test the hypothesis that switching endocrine agents at approximately 2 years from randomization for patients who are disease-free is superior to continuing with the original agent. RESULTS: The 2005 analysis showed the superiority of letrozole compared with tamoxifen. The patients who were assigned tamoxifen alone were unblinded and offered the opportunity to switch to letrozole. Results from other trials increased the clinical relevance about whether or not to start treatment with letrozole or tamoxifen, and analysis plans were expanded to evaluate sequential versus single-agent strategies from randomization. LIMITATIONS: Due to the unblinding of patients assigned tamoxifen alone, analysis of updated data will require ascertainment of the influence of selective crossover from tamoxifen to letrozole. CONCLUSIONS: BIG 1-98 is an example of an enriched design, involving complementary analyses addressing different questions several years apart, and subject to evolving analytic plans influenced by new data that emerge over time.

A validated assay by liquid chromatography-tandem mass spectrometry for the simultaneous quantification of elvitegravir and rilpivirine in HIV positive patients.

Because of the large variability in the pharmacokinetics of anti-HIV drugs, therapeutic drug monitoring in patients may contribute to optimize the overall efficacy and safety of antiretroviral therapy. An LC-MS/MS method for the simultaneous assay in plasma of the novel antiretroviral agents rilpivirine (RPV) and elvitegravir (EVG) has been developed to that endeavor. Plasma samples (100 μL) extraction is performed by protein precipitation with acetonitrile, and the supernatant is subsequently diluted 1:1 with 20-mM ammonium acetate/MeOH 50:50. After reverse-phase chromatography, quantification of RPV and EVG, using matrix-matched calibration samples, is performed by electrospray ionization-triple quadrupole mass spectrometry by selected reaction monitoring detection using the positive mode. The stable isotopic-labeled compounds RPV-(13) C6 and EVG-D6 were used as internal standards. The method was validated according to FDA recommendations, including assessment of extraction yield, matrix effects variability (<6.4%), as well as EVG and RPV short and long-term stability in plasma. Calibration curves were validated over the clinically relevant concentrations ranging from 5 to 2500 ng/ml for RPV and from 50 to 5000 ng/ml for EVG. The method is precise (inter-day CV%: 3-6.3%) and accurate (3.8-7.2%). Plasma samples were found to be stable (<15%) in all considered conditions (RT/48 h, +4°C/48 h, -20°C/3 months and 60°C/1 h). Selected metabolite profiles analysis in patients' samples revealed the presence of EVG glucuronide, that was well separated from parent EVG, allowing to exclude potential interferences through the in-source dissociation of glucuronide to parent drug. This new, rapid and robust LCMS/MS assay for the simultaneous quantification of plasma concentrations of these two major new anti-HIV drugs EVG and RPV offers an efficient analytical tool for clinical pharmacokinetics studies and routine therapeutic drug monitoring service. Copyright © 2013 John Wiley & Sons, Ltd.

High-magnification vascular imaging to reject false-positive sites in situ during Hexvix® fluorescence cystoscopy

Fluorescence imaging for detection of non-muscle-invasive bladder cancer is based on the selective production and accumulation of fluorescing porphyrins-mainly, protoporphyrin IX-in cancerous tissues after the instillation of Hexvix®. Although the sensitivity of this procedure is very good, its specificity is somewhat limited due to fluorescence false-positive sites. Consequently, magnification cystoscopy has been investigated in order to discriminate false from true fluorescence positive findings. Both white-light and fluorescence modes are possible with the magnification cystoscope, allowing observation of the bladder wall with magnification ranging between 30× for standard observation and 650×. The optical zooming setup allows adjusting the magnification continuously in situ. In the high-magnification (HM) regime, the smallest diameter of the field of view is 600 microns and the resolution is 2.5 microns when in contact with the bladder wall. With this cystoscope, we characterized the superficial vascularization of the fluorescing sites in order to discriminate cancerous from noncancerous tissues. This procedure allowed us to establish a classification based on observed vascular patterns. Seventy-two patients subject to Hexvix® fluorescence cystoscopy were included in the study. Comparison of HM cystoscopy classification with histopathology results confirmed 32/33 (97%) cancerous biopsies and rejected 17/20 (85%) noncancerous lesions.

N-Glycans on secretory component: mediators of the interaction between secretory IgA and gram-positive commensals sustaining intestinal homeostasis.

Human beings live in symbiosis with billions of microorganisms colonizing mucosal surfaces. The understanding of the mechanisms underlying this fine-tuned intestinal balance has made significant processes during the last decades. We have recently demonstrated that the interaction of SIgA with Gram-positive bacteria is essentially based on Fab-independent, glycan-mediated recognition. Results obtained using mouse hybridoma- and colostrum-derived secretory IgA (SIgA) consistently show that N-glycans present on secretory component (SC) play a crucial role in the process. Natural coating may involve specific Gram-positive cell wall components, which may explain selective recognition at the molecular level. More widely, the existence of these complexes is involved in the modulation of intestinal epithelial cell (IEC) responses in vitro and the formation of intestinal biofilms. Thus, SIgA may act as one of the pillars in homeostatic maintenance of the microbiota in the gut, adding yet another facet to its multiple roles in the mucosal environment.

Evaluation of the compliance with recommended procedures in newborns exposed to HBsAg-positive mothers: a multicenter collaborative study.

BACKGROUND: Maternal-infant transmission of hepatitis B virus (HBV) during birth carries a high risk for chronic HBV infection in infants with frequent subsequent development of chronic disease. This can be efficiently prevented by early immunization of exposed newborns. The purpose of this study was to determine the compliance with official recommendations for prevention of perinatal HBV transmission in hepatitis B surface antigen (HBsAg) exposed infants. METHODS: Records of pregnant women at 4 sites in Switzerland, admitted for delivery in 2005 and 2006, were screened for maternal HBsAg testing. In HBsAg-exposed infants, recommended procedures (postnatal active and passive immunization, completion of immunization series, and serological success control) were checked. RESULTS: Of 27,131 women tested for HBsAg, 194 (0.73%) were positive with 196 exposed neonates. Of these neonates, 143 (73%) were enrolled and 141 (99%) received simultaneous active and passive HBV immunization within 24 hours of birth. After discharge, the HBV immunization series was completed in 83%. Only 38% of children were tested for anti-HBs afterwards and protective antibody values (>100 U/L) were documented in 27% of the study cohort. No chronically infected child was identified. Analysis of hospital discharge letters revealed significant quality problems. CONCLUSIONS: Intensified efforts are needed to improve the currently suboptimal medical care in HBsAg-exposed infants. We propose standardized discharge letters, as well as reminders to primary care physicians with precise instructions on the need to complete the immunization series in HBsAg-exposed infants and to evaluate success by determination of anti-HBs antibodies after the last dose.

Is chemotherapy necessary for premenopausal women with lower-risk node-positive, endocrine responsive breast cancer? 10-year update of International Breast Cancer Study Group Trial 11-93.

INTRODUCTION: International Breast Cancer Study Group (IBCSG) Trial 11-93 is the largest trial evaluating the role of the addition of chemotherapy to ovarian function suppression/ablation (OFS) and tamoxifen in premenopausal patients with endocrine-responsive early breast cancer. METHODS: IBCSG Trial 11-93 is a randomized trial comparing four cycles of adjuvant chemotherapy (AC: doxorubicin or epirubicin, plus cyclophosphamide) added to OFS and 5 years of tamoxifen versus OFS and tamoxifen without chemotherapy in premenopausal patients with node-positive, endocrine-responsive early breast cancer. There were 174 patients randomized from May 1993 to November 1998. The trial was closed before the target accrual was reached due to low accrual rate. RESULTS: Patients randomized tended to have lower risk node-positive disease and the median age was 45. After 10 years median follow up, there remains no difference between the two randomized treatment groups for disease-free (hazard ratio=1.02 (0.57-1.83); P=0.94) or overall survival (hazard ratio=0.97 (0.44-2.16); P=0.94). CONCLUSION: This trial, although small, offers no evidence that AC chemotherapy provides additional disease control for premenopausal patients with lower-risk node-positive endocrine-responsive breast cancer who receive adequate adjuvant endocrine therapy. A large trial is needed to determine whether chemotherapy adds benefit to endocrine therapy for this population.

Sublimation of new matrix candidates for high spatial resolution imaging mass spectrometry of lipids: enhanced information in both positive and negative polarities after 1,5-diaminonapthalene deposition.

Matrix sublimation has demonstrated to be a powerful approach for high-resolution matrix-assisted laser desorption ionization (MALDI) imaging of lipids, providing very homogeneous solvent-free deposition. This work presents a comprehensive study aiming to evaluate current and novel matrix candidates for high spatial resolution MALDI imaging mass spectrometry of lipids from tissue section after deposition by sublimation. For this purpose, 12 matrices including 2,5-dihydroxybenzoic acid (DHB), sinapinic acid (SA), α-cyano-4-hydroxycinnamic acid (CHCA), 2,6-dihydroxyacetphenone (DHA), 2',4',6'-trihydroxyacetophenone (THAP), 3-hydroxypicolinic acid (3-HPA), 1,8-bis(dimethylamino)naphthalene (DMAN), 1,8,9-anthracentriol (DIT), 1,5-diaminonapthalene (DAN), p-nitroaniline (NIT), 9-aminoacridine (9-AA), and 2-mercaptobenzothiazole (MBT) were investigated for lipid detection efficiency in both positive and negative ionization modes, matrix interferences, and stability under vacuum. For the most relevant matrices, ion maps of the different lipid species were obtained from tissue sections at high spatial resolution and the detected peaks were characterized by matrix-assisted laser desorption ionization time-of-flight/time-of-flight (MALDI-TOF/TOF) mass spectrometry. First proposed for imaging mass spectrometry (IMS) after sublimation, DAN has demonstrated to be of high efficiency providing rich lipid signatures in both positive and negative polarities with high vacuum stability and sub-20 μm resolution capacity. Ion images from adult mouse brain were generated with a 10 μm scanning resolution. Furthermore, ion images from adult mouse brain and whole-body fish tissue sections were also acquired in both polarity modes from the same tissue section at 100 μm spatial resolution. Sublimation of DAN represents an interesting approach to improve information with respect to currently employed matrices providing a deeper analysis of the lipidome by IMS.

Empirical use of anti-Gram-positive antibiotics in febrile neutropaenic cancer patients with acute leukaemia

Gram-positive infections including those due to methicillin-resistant staphylococci occur frequently in febrile neutropaenic patients. Although few data support the empirical addition of a glycopeptide antibiotic to the standard broad-spectrum antibiotic regimen, these agents are often used in many cancer centres. The emergence of infections due to vancomycin- resistant enterococci and glycopeptide-intermediate staphylococci has led to recommendations for a restricted use of glycopeptide antibiotics. The objective of the present work was to formulate evidence-based guidelines for the empirical use of anti- Gram-positive antibiotics in neutropaenic patients with acute leukaemia.

An epidemiologic study to determine the prevalence of the HLA-B*5701 allele among HIV-positive patients in Europe.

OBJECTIVES: HLA-B*5701 is a major histocompatibility complex class I allele associated with an immunologically-mediated hypersensitivity reaction to abacavir. The objectives of this study were to evaluate HLA-B*5701 prevalence among European, HIV-1-infected patients and to compare the local and central laboratory screening results. METHODS: Data were combined from six multicentre, prospective studies involving 10 European countries in which HIV-1-infected patients (irrespective of treatment experience or previous HLA-B*5701 screening), >or=18 years of age, were evaluated for HLA-B*5701 carriage, determined by the central and local laboratory methods. RESULTS: A total of 9720 patients from 272 centres were included in the analysis. The overall estimate of HLA-B*5701 prevalence in Europe was 4.98%, with country-specific estimates ranging from 1.53 to 7.75%. HLA-B*5701 prevalence was highest in the self-reported white population (6.49%) and lowest in the black population (0.39%). Local laboratory results had a high specificity (99.9%) and sensitivity (99.2%) when compared with the central laboratory results. CONCLUSION: This study supports data from previous studies regarding the prevalence of HLA-B*5701 in the HIV population and the variation of HLA-B*5701 prevalence between different racial groups. The high specificity and sensitivity of local laboratory results, suggests that clinicians can be confident in using local laboratories for pretreatment HLA-B*5701 screening. However, it is essential that local laboratories participate in HLA-B*5701-specific quality assurance programs to maintain 100% sensitivity. In HIV-infected patients, pretreatment HLA-B*5701 screening may allow more informed decisions regarding abacavir use and has the potential to significantly reduce the frequency of abacavir-related hypersensitivity reactions and costs associated with managing these reactions.