Explaining survival differences between two consecutive studies with allogeneic stem cell transplantation in patients with chronic myeloid leukemia

Purpose

In the two consecutive German studies III and IIIA on chronic myeloid leukemia, between 1995 and 2004, 781 patients were randomized to receive either allogeneic hematopoietic stem cell transplantation with a related donor or continued drug treatment. Despite comparable transplantation protocols and most centers participating in both studies, the post-transplant survival probabilities for patients transplanted in first chronic phase were significantly higher in study IIIA (144 patients) than in study III (113 patients). Prior to the decision on a combined analysis of both studies, reasons for this discrepancy had to be investigated.

Methods

The Cox proportional hazard cure model was used to identify prognostic factors for post-transplant survival.

Results

Donor–recipient matching for human leukocyte antigen, patient age, time between diagnosis and transplantation, and calendar time showed a significant influence on survival and/or the incidence of cure. Added as a further factor, affiliation to study IIIA had no significant impact any longer.

Conclusions

Discrepancies in influential prognostic factors explained the different post-transplant survival probabilities between the studies. The significance of calendar time suggests a lack of consistency of transplantation practice over time. Accordingly, the prerequisite for a common assessment of overall survival in the two randomized transplantation arms was not met. Moreover, our analyses provide an independent validation of established prognostic factors and their cutoffs. The statistical approach in investigating and modeling potential prognostic factors for survival sets an example for the examination of studies with unexpected outcome differences in concurrent treatment arms.     

Echocardiographic Changes in Patients Implanted With a Fully Magnetically Levitated Left Ventricular Assist Device (Heartmate 3)

Background

The Heartmate 3 (HM3) is a Conformiteé Européenne mark–approved left ventricular (LV) assist device (LVAD) with fully magnetically levitated rotor and features consisting of a wide range operational speeds, wide flow paths, and artificial pulse. We performed a hemodynamic-echocardiographic speed optimization evaluation in HM3-implanted patients to achieve optimal LV- and right ventricular (RV) shape.

Subtypes of autism by cluster analysis based on structural MRI data

Abstract The aim of our study was to subcategorize Autistic Spectrum Disorders (ASD) using a multidisciplinary approach. Sixty four autistic patients (mean age 9.4±5.6 years) were entered into a cluster analysis. The clustering analysis was based on MRI data. The clusters obtained did not differ significantly in the overall severity of autistic symptomatology as measured by the total score on the Childhood Autism Rating Scale (CARS). The clusters could be characterized as showing significant differences: Cluster 1: showed the largest sizes of the genu and splenium of the corpus callosum (CC), the lowest pregnancy order and the lowest frequency of facial dysmorphic features. Cluster 2: showed the largest sizes of the amygdala and hippocampus (HPC), the least abnormal visual response on the CARS, the lowest frequency of epilepsy and the least frequent abnormal psychomotor development during the first year of life. Cluster 3: showed the largest sizes of the caput of the nucleus caudatus (NC), the smallest sizes of the HPC and facial dysmorphic features were always present. Cluster 4: showed the smallest sizes of the genu and splenium of the CC, as well as the amygdala, and caput of the NC, the most abnormal visual response on the CARS, the highest frequency of epilepsy, the highest pregnancy order, abnormal psychomotor development during the first year of life was always present and facial dysmorphic features were always present. This multidisciplinary approach seems to be a promising method for subtyping autism.     

Efficiency of acidemia correction on intermittent versus continuous hemodialysis in acute methanol poisoning

Context: Acidemia is a marker of prognosis in methanol poisoning, as well as compounding formate-induced cytotoxicity. Prompt correction of acidemia is a key treatment of methanol toxicity and methods to optimize this are poorly defined.

Objective: We studied the efficiency of acidemia correction by intermittent hemodialysis (IHD) and continuous renal replacement therapy (CRRT) in a mass outbreak of methanol poisoning.

Methods: The study was designed as observational cohort study. The mean time for an increase of 1?mmol/L HCO₃^–, 0.01 unit arterial blood pH, and the total time for correction of HCO₃^– were determined in IHD- and CRRT-treated patients.

Results: Data were obtained from 18 patients treated with IHD and 13 patients treated with CRRT. At baseline, CRRT group was more acidemic than IHD group (mean arterial pH 6.79?±?0.10 versus 7.05?±?0.10; p?=?0.001). No association was found between the rate of acidemia correction and age, weight, serum methanol, lactate, formate, and glucose on admission. The time to HCO₃^– correction correlated with arterial blood pH (r=??0.511; p?=?0.003) and creatinine (r?=?0.415; p?=?0.020). There was association between the time to HCO₃^– correction and dialysate/effluent and blood flow rates (r=??0.738; p?r=??0.602; p?The mean time for HCO₃^– to increase by 1?mmol/L was 12?±?2?min for IHD versus 34?±?8?min for CRRT (p?p?=?0.024). The mean increase in HCO₃^– was 5.67?±?0.90?mmol/L/h for IHD versus 2.17?±?0.74?mmol/L/h for CRRT (p?Conclusions: Our study supports the superiority of IHD over CRRT in terms of the rate of acidemia correction.     

Deregulation of the G1/S-phase control in human testicular germ cell tumours

Deregulated cell cycle and defective genome-integrity checkpoints are among the hallmarks of cancer.Here we summarize our recent studies of key components of the GI/S machinery in normal human spermatogenesis, and their abnormalities in testicular germ cell tumours (TGCTs), with special emphasis on carcinoma in situ lesions (CIS). Our combined immunohistochemical and immunoblotting analyses of normal human adult and fetal testes, CIS, seminomas, embryonal carcinomas, and teratomas, revealed an 'unorthodox' spectrum of defects within the so-called RB pathway in TGCTs. The early aberrations included lack of expression of the retinoblastoma tumour suppressor (pRB) and the CDK inhibitor pl9ink4d, and overexpression of cyclin D2. Progression from CIS to invasive TGCTswas associated with loss of another two CDK inhibitors and tumour suppressors: pl6ink4a and pl8ink4c. We also found the lack of pRB and pl9ink4d in fetal gonocytes, the candidate target cell for all types of TGCTs. These findings, together with the status of the Chk2-p53 DNA-integrity checkpoint, are considered in relation to the origin, biology and pathogenesis of TGCTs, and potential implications of the GI/S defects for the curability of these tumours.     

Rapid Detection and Identification of Mucormycetes from Culture and Tissue Samples by Use of High-Resolution Melt Analysis

We present a method for rapid and simple detection of clinically relevant mucormycetes of the Mucorales order in cultures and clinical samples. This seminested real-time PCR uses mucormycete-specific primers and is followed by species identification using high-resolution melt (HRM) analysis. The method is highly suitable for routine clinical diagnostics.Invasive infections caused by mucormycetes started to occur more frequently in the last decade and are connected with rapid progression and high mortality rates. Early diagnostics and targeted treatment are crucial. Most mucormycosis cases (over 90%) are caused by Rhizopus spp., followed by Mucor spp., Lichtheimia spp., Rhizomucor pusillus, and, rarely, some other species (2, 9, 11, 16).Definitive diagnosis of mucormycosis is usually made after histopathological proof of mucormycete-like hyphae in involved tissue; the causative agent can be determined only by culture (13). So far, no serological test is available and radiological methods are nonspecific.Molecular detection of mucormycetes is complicated by several factors, and we still do not have any standard protocol. Few methods for the detection of mucormycetes have been published, and only some have been evaluated using clinical samples (1, 5, 10, 14, 15, 17) or samples from animal models (6, 7).The aim of this study was to develop a rapid and sensitive technique for the detection and identification of clinically important mucormycetes. We adopted primers from a qualitative method previously published by Bialek et al. (1) that is specific for members of the order Mucorales targeting 18S ribosomal DNA (rDNA). We modified it to seminested real-time PCR with EvaGreen dye, followed by species distinction by high-resolution melt (HRM) analysis. HRM analysis uses amplification of DNA in the presence of intercalation dye. Fluorescence is measured during a controlled melting of PCR product that results in a melt curve that depends mainly on GC content, length, and sequence of the PCR product. This simple method can be used for genotyping or mutation scanning without the need for time-consuming sequencing (4, 12).DNA was isolated from 50 μl of fungal culture (inoculum was prepared by covering sporulating colonies with approximately 2 ml of sterile 0.85% saline) or a piece of fresh tissue (2 by 1 mm) using the ZR fungal/bacterial DNA kit (Zymo Research). Tissue samples were incubated in lysis buffer overnight, and cultures were immediately processed according to the manufacturer''s protocol. Disruption was extended to 15 min (Disruptor Genie; Scientific Industries). DNA from formalin-fixed, paraffin wax-embedded (FFPE) tissue samples was isolated from 2 or 3 scrolls (5 to 10 μm each) of paraffin block using a DNeasy blood and tissue kit (Qiagen). Paraffin was dissolved in 1 ml of xylene, and then the tissue was washed two times using 1 ml of 96% ethanol and incubated in 180 μl of ATL buffer (Qiagen) and 20 μl of proteinase K (600 mAU/ml solution, where one mAU represents the activity of proteinase K that releases folin-positive amino acids and peptides corresponding to 1 μmol of tyrosine per min) at 55°C overnight and then at 90°C for 1 h. The next steps were done in accordance with the manufacturer''s protocol. DNA isolation from clinical samples was done in a biological safety cabinet. An aliquot of sterile water was processed with each set of samples as a control of potential contamination during the isolation process.Five microliters of DNA was amplified in 25 μl of amplification mixture that contained a 0.2 μM concentration each of primers ZM1 and ZM2 (1), 120 μM deoxynucleoside triphosphates (dNTPs; Roche, Germany), 2.5 mM MgCl₂, 1× GeneAmp PCR Gold buffer, and 1.5 U AmpliTaq Gold DNA polymerase (Applied Biosystems). The cycling conditions were 10 min at 95°C, 16 cycles of 30 s at 94°C, 30 s at 50°C, and 60 s at 72°C, and 7 min at 72°C. One microliter of PCR product from the external round was then amplified in duplicate using Rotorgene 6000 (Corbett Research, Australia). Twenty-five microliters of the amplification mixture contained a 0.4 μM concentration each of primers ZM1 and ZM3 (1), 12.5 μl of SensiMix HRM, and 1 μl of EvaGreen (both from a SensiMix HRM kit; Quantace, United Kingdom). The cycling conditions were 10 min at 95°C, followed by 40 cycles of 15 s at 95°C, 20 s at 60°C, and 30 s at 72°C (acquired on the green channel), followed by HRM analysis (ramp from 74°C to 79.5°C, rising by 0.1°C each cycle, acquired on the HRM channel). Rotorgene 6000 series software (version 1.7) was used for analysis of the results. All positive results were confirmed by sequencing of the PCR product. DNA was purified using a QIAquick PCR purification kit (Qiagen, Germany) and sequenced using a BigDye Terminator v1.1 cycle sequencing kit (Applied Biosystems) on an ABI Prism 310 genetic analyzer (Applied Biosystems). Sequences were analyzed using the BLAST alignment program of the GenBank database.We used DNA extracted from five mucormycete cultures diluted in Tris-EDTA (TE) buffer as positive controls in every run. A DNA isolation control (sterile water processed with clinical samples) and a negative control of PCR (sterile water) were added to each run as well.In this study, we tested 31 fungal isolates, comprising 10 mucormycete isolates and 21 isolates from other filamentous fungal groups (Department of Clinical Microbiology, University Hospital Brno and Czech Collection of Microorganisms, Czech Republic). All mucormycete isolates were correctly identified. The melting temperatures (T_m) for each species were as follows: for Rhizopus microsporus, 76.46°C; for Rhizopus oryzae, 76.59°C; for Mucor racemosus, 76.78°C; for Mucor circinelloides, 76.98°C; for Rhizomucor pusillus, 77.87°C; and for Lichtheimia corymbifera, 78.56°C. Representative HRM curves for six different mucormycetes are shown in Fig. ​Fig.1.1. All HRM analysis results were confirmed by sequencing. None of the nonmucormycete fungi were positively tested. The results are summarized in Table ​Table11.Open in a separate windowFIG. 1.Representative result of high-resolution melt (HRM) analysis. Shown are HRM curves for six mucormycete isolates (black curves) and one negative and one positive tissue sample (gray curves).

TABLE 1.

List of fungal isolates used in this study and results of HRM analysis^a

Influenza virus infection of the murine uterus: a new model for antiviral immunity in the female reproductive tract

Secretory IgA (S-IgA) mediates local immunity to influenza virus in the murine upper respiratory tract and may play an important role in local immunity to various microorganisms in the female reproductive tract as well. Although the presence of IgA in cervicovaginal or uterine secretions has been correlated with immunity to a number of pathogens, there has been no direct demonstration of the mediation of uterine antiviral immunity by S-IgA. Influenza virus, although not a normal pathogen of the reproductive tract, was used to develop a model for the investigation of mucosal immunity in the uterus. PR8 (H1N1) influenza virus injected into the ovarian bursa of BALB/c mice grew well, with peak titers between days 3 and 5. Intravenous injection of polymeric IgA anti-influenza virus monoclonal antibody before or 30 min after viral challenge protected mice against viral infection. We believe this work to be the first direct demonstration of S-IgA-mediated antiviral uterine immunity. It provides a model for further investigation of immunity in the female reproductive tract.