Analysing time to event data in dementia prevention trials: The example of the guidage study of EGB761®

Time-to-event analysis is frequently used in medical research to investigate potential diseasemodifying treatments in neurodegenerative diseases. Potential treatment effects are generally evaluated using the logrank test, which has optimal power and sensitivity when the treatment effect (hazard ratio) is constant over time. However, there is generally no prior information as to how the hazard ratio for the event of interest actually evolves. In these cases, the logrank test is not necessarily the most appropriate to use. When the hazard ratio is expected to decrease or increase over time, alternative statistical tests such as the Fleming-Harrington test, provide a better sensitivity. An example of this comes from a large, five-year randomised, placebo-controlled prevention trial (GuidAge) in 2854 community-based subjects making spontaneous memory complaints to their family physicians, which evaluated whether treatment with EGb761® can modify the risk of developing AD. The primary outcome measure was the time to conversion from memory complaint to Alzheimer’s type dementia. Although there was no significant difference in the hazard function of conversion between the two treatment groups according to the preplanned logrank test, a significant treatment-by-time interaction for the incidence of AD was observed in a protocol-specified subgroup analysis, suggesting that the hazard ratio is not constant over time. For this reason, additional post hoc analyses were performed using the Fleming-Harrington test to evaluate whether there was a signal of a late effect of EGb761®. Applying the Fleming-Harrington test, the hazard function for conversion to dementia in the placebo group was significantly different from that in the EGb761® treatment group (p = 0.0054), suggesting a late effect of EGb761®. Since this was a post hoc analysis, no definitive conclusions can be drawn as to the effectiveness of the treatment. This post hoc analysis illustrates the interest of performing another randomised clinical trial of EGb761® explicitly testing the hypothesis of a late treatment effect, as well as of using of better adapted statistical approaches for long term preventive trials when it is expected that prevention cannot have an immediate effect but rather a delayed effect that increases over time.     

Front-line high-dose therapy with autologous stem cell transplantation for high risk Hodgkin's disease: comparison with combined-modality therapy

This retrospective study compares high-dose therapy (HDT) with autologous stem cell transplantation and combined-modality treatment (CT) as a first-line therapy for Hodgkin's disease (HD) for patients with both a clinical stage (CS) IV and/or a mediastinal mass > or =0.45 of the thoracic diameter (MM > or =0.45) at diagnosis, and an incomplete response after the first-line chemotherapy. Data on 42 grafted patients (GP) in Nantes Hospital, France and on 108 combined-modality treated patients (CTP) from two protocols of the GOELAMS group, France (POF 81 and H90) was analyzed. Both groups were comparable except for pulmonary disease in excess in the grafted group (P = 0.01). Among GP, 95% were in complete response at the end of first-line treatment and 77% among CTP. Median follow-up was 53 months (range, 7 to 128 months) for GP and 88 months (range, 25 to 181 months) for CTP. The 5-year freedom from progression (FFP) and event-free survival (EFS) rates were better for GP (87% vs 55% for FFP: P = 0.0004 and 81% vs 51% for EFS: P = 0.0004) whereas the overall survival (OS) rates did not differ significantly (85% for GP vs 71% for CTP: P = 0.06). Similar results were obtained for the groups with a response > or =50% after initial chemotherapy: 91% vs 65% for FFP, P = 0.01; 87% vs 61% for EFS, P = 0.02; and 92% vs 77% for OS, P = 0.2; and for the groups with a response <50%: 80% vs 22% for FFP, P = 0.0003; 72% vs 13% for EFS, P = 0.0001; and 76% vs 46% for OS, P = 0.04. This study shows a better control of the disease with HDT.     

Estimation of the familial relative risk of cancer by site from a French population based family study on colorectal cancer (CCREF study)

BACKGROUND: Colorectal cancer (CRC) is the second most common cause of death from cancer in France. A family history of CRC increases an individual's risk of developing CRC. Family history has been suggested to have a greater impact on proximal than distal tumours. AIM: We estimated the familial risk of CRC and other cancers, and examined how risk varies according to localisation of the tumour in the colorectal tract. SUBJECTS: We recorded all cases of CRC diagnosed between 1993 and 1998 in the region served by the Calvados Cancer Registry. A trained interviewer asked all participants about their family history of cancer. STATISTICAL METHODS: Familial risk was estimated from a cohort analysis of the relatives of the CRC cases. The expected numbers of cancers were calculated from Calvados incidence rates. Familial relative risks were calculated using standardised incidence ratios. RESULTS: Our findings showed that colon cancer had a stronger familial/genetic component (relative risk (RR) 1.47) than rectal cancer (RR 0.98). The familial/genetic component appeared stronger for proximal colon cancer than for distal colon cancer only among women (RR 2.24 v RR 1.45). CRC appeared to be positively associated with leukaemia (RR 1.77), stomach cancer (RR 1.32), and testicular cancer (RR 3.13), and negatively associated with urinary bladder cancer (RR 0.57) within families. The cancer spectrum associated with CRC among younger participants included prostate (RR 1.93), uterus (RR 2.49), and thyroid (RR 3.85) cancers. CONCLUSION: If our results are confirmed, follow up guidelines for patients with a family history of CRC should depend on the sex and tumour site of affected relatives to avoid needless invasive screening.     

Amplification of genes encoding human myeloid membrane antigens after DNA-mediated gene transfer

Spontaneous amplification of genes encoding two different human myeloid surface antigens was observed after DNA-mediated gene transfer of cellular DNA from the human myeloid cell line HL-60 into NIH-3T3 mouse fibroblasts. Transformed recipient cells with highly amplified expression of either of two donor membrane polypeptides, gp150 or p67, were isolated with a fluorescence-activated cell sorter (FACS), using monoclonal antibodies specific for human myeloid cells. Immunoprecipitation of enzymatically radioiodinated polypeptides from the surface of transformed NIH-3T3 cells confirmed that expression of these proteins was amplified tenfold to 20-fold in comparison to their expression on human myeloid cell lines. The cellular DNA of cloned secondary and tertiary transformants expressing high levels of gp150 and p67 contained amplified sets of DNA restriction fragments that hybridized with human repetitive DNA sequences. Cytogenetic analysis of subclones overexpressing gp150 revealed extrachromosomal double minutes (DMs), whose presence correlated with the unstable expression of the membrane polypeptide. Human sequences in gp150-positive clones did not localize to chromosomes, consistent with their association with extrachromosomal DMs. By contrast, p67-positive subclones stably expressed the antigen, and in situ hybridization to metaphase spreads demonstrated that amplified human DNA sequences were integrated into a specific marker chromosome. Cytogenetic analysis of the parental NIH- 3T3 subclone used in these studies disclosed DMs in a low percentage of metaphases, suggesting that the recipient cells have a propensity for amplifying donor DNA.     

Molecular heterogeneity in acute leukemia lineage switch

Six cases of acute leukemia that underwent lineage switch from acute lymphocytic leukemia to acute myelogenous leukemia are reported. The mean age of the patients was 24 years, time to conversion was 36 months, and survival after conversion was only 3 months. Of the three cases which showed abnormal metaphases at both diagnosis and conversion, two (cases 2, 5) showed related cytogenetic abnormalities, and the third showed (case 3) independent chromosomal changes. Molecular analysis for immunoglobulin heavy chain and T-cell receptor beta chain genes showed that five of the six cases had rearrangement of at least one of these lymphoid associated genes at conversion to acute myelogenous leukemia. The single case (case 3) in which there were no lymphoid gene rearrangements at conversion was also the only case in which independent karyotypic abnormalities at diagnosis and conversion were demonstrated. Our findings suggest that lineage switch can represent either relapse of the original clone with heterogeneity at the molecular level or the emergence of a second new leukemic clone without molecular heterogeneity.     

Biological evaluation of intervertebral disc cells in different formulations of gellan gum‐based hydrogels

Gellan gum (GG)‐based hydrogels are advantageous in tissue engineering not only due to their ability to retain large quantities of water and provide a similar environment to that of natural extracellular matrix (ECM), but also because they can gelify in situ in seconds. Their mechanical properties can be fine‐tuned to mimic natural tissues such as the nucleus pulposus (NP). This study produced different formulations of GG hydrogels by mixing varying amounts of methacrylated (GG‐MA) and high‐acyl gellan gums (HA‐GG) for applications as acellular and cellular NP substitutes. The hydrogels were physicochemically characterized by dynamic mechanical analysis. Degradation and swelling abilities were assessed by soaking in a phosphate buffered saline solution for up to 170 h. Results showed that as HA‐GG content increased, the modulus of the hydrogels decreased. Moreover, increases in HA‐GG content induced greater weight loss in the GG‐MA/HA‐GG formulation compared to GG‐MA hydrogel. Potential cytotoxicity of the hydrogel was assessed by culturing rabbit NP cells up to 7 days. An MTS assay was performed by seeding rabbit NP cells onto the surface of 3D hydrogel disc formulations. Viability of rabbit NP cells encapsulated within the different hydrogel formulations was also evaluated by Calcein‐AM and ATP assays. Results showed that tunable GG‐MA/HA‐GG hydrogels were non‐cytotoxic and supported viability of rabbit NP cells. Copyright © 2012 John Wiley & Sons, Ltd.     

High resolution of heterogeneity among human neutrophil granules: physical, biochemical, and ultrastructural properties of isolated fractions

Previous studies on the fractionation of human neutrophil granules have identified two major populations: myeloperoxidase (MPO)-containing azurophil, or primary, granules and MPO-deficient specific, or secondary, granules. Peripheral blood neutrophils from individual donors were lysed in sucrose-free media by either hypotonic shock or nitrogen cavitation. Using a novel two-gradient Percoll density centrifugation system, the granule-rich postnuclear supernatant was rapidly (ten minutes) and reproducibly resolved into 13 granule fractions (L1 through L8 and H1 through H5). Granule flotation and recentrifugation experiments on both continuous, self-generated and multiple-step gradients using individual and mixed isolated fractions demonstrated that the banding patterns were isopycnic and nonartifactual. Isolated granules were intact based on the findings that biochemical latency of several granule enzymes was greater than 95%, and thin-sectioned electron micrographs demonstrated intact granule profiles. Biochemical analyses of the granule marker proteins MPO, beta-glucuronidase, lysozyme, and lactoferrin indicated that a number of the fractions were related to the major azurophil and specific granule populations. Lactoferrin was found in ten of 13 fractions (L1 through L8, H1 to H2), whereas MPO was found in every fraction. Consistent with these biochemical data, all fractions exhibited varying degrees of heterogeneity based on ultrastructural morphology and cytochemistry, including diaminobenzidine (DAB) reactivity for peroxidase and periodate-thiocarbohydrazide-silver proteinate (PA-TCH-SP) staining for complex glycoconjugates. A variable but significant percentage (23% to 70%) of the granules in fractions L1 through L8 and H1 and H2 showed DAB reactivity, while about 90% of the granules in fractions H3 through H5 were peroxidase positive. These results demonstrated that DAB-reactive granules spanned the entire range of granule size and density. Ultrastructural PA-TCH-SP staining of isolated granule fractions revealed patterns similar to those of granules in intact neutrophils at different stages of development. Granules from human acute promyelocytic leukemia cells (HL-60) exhibited a surprisingly low density compared with typical azurophil granules from normal, mature neutrophils. The data suggest that both functional and maturational differences contribute to granule heterogeneity, and provide a new practical and conceptual framework for further defining the phenomenon of neutrophil granule heterogeneity.     

Evaluation of lockdown effect on SARS-CoV-2 dynamics through viral genome quantification in waste water,Greater Paris,France, 5 March to 23 April 2020

IntroductionSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the etiological agent of coronavirus disease (COVID-19). People infected with SARS-CoV-2 may exhibit no or mild non-specific symptoms; thus, they may contribute to silent circulation of the virus among humans. Since SARS-CoV-2 RNA can be detected in stool samples, monitoring SARS-CoV-2 RNA in waste water (WW) has been proposed as a complementary tool to investigate virus circulation in human populations.AimTo test if the quantification of SARS-CoV-2 genomes in WW correlates with the number of symptomatic or non-symptomatic carriers.MethodWe performed a time-course quantitative analysis of SARS-CoV-2 by RT-qPCR in raw WW samples collected from several major WW treatment plants in Greater Paris. The study period was 5 March to 23 April 2020, including the lockdown period in France (from 17 March).ResultsWe showed that the increase of genome units in raw WW accurately followed the increase of human COVID-19 cases observed at the regional level. Of note, the viral genome could be detected before the epidemic grew massively (around 8 March). Equally importantly, a marked decrease in the quantities of genome units was observed concomitantly with the reduction in the number of new COVID-19 cases, 29 days following the lockdown.ConclusionThis work suggests that a quantitative monitoring of SARS-CoV-2 genomes in WW could generate important additional information for improved monitoring of SARS-CoV-2 circulation at local or regional levels and emphasises the role of WW-based epidemiology.