Monitoring of adenovirus load in stool by real-time PCR permits early detection of impending invasive infection in patients after allogeneic stem cell transplantation.

Invasive adenovirus (AdV) infections are associated with high morbidity and mortality in allogeneic stem cell transplant recipients. We observed that molecular detection of the virus in stool specimens commonly precedes AdV viremia, suggesting that intestinal infections may represent a common source of virus dissemination. To address this notion, we have investigated 153 consecutive allogeneic transplantations in 138 pediatric patients by quantitative monitoring of AdV in stool specimens and peripheral blood by a pan-adenovirus real-time (RQ)-PCR approach. AdV was detectable in serial stool specimens in all cases of AdV viremia during the post-transplant course (P<0.0001). The incidence of AdV viremia in individuals with peak virus levels in stool specimens above 1 x 10E6 copies per gram (n=22) was 73% vs 0% in patients with AdV levels in stool specimens below this threshold (n=29; P<0.0001). Serial measurement of AdV levels in stool specimens by RQ-PCR permitted early diagnosis of impending invasive infection with a sensitivity and specificity of 100% (95% confidence interval (CI) 96-100%) and 83% (95% CI 67-92%), respectively. The median time span between detection of AdV loads in stool specimens above 1 x 10E6 copies per gram and first observation of viremia was 11 days (range 0-192). Quantitative monitoring of the AdV load in stool specimens therefore provides a rationale for early initiation of antiviral treatment with the aim of preventing progression to life-threatening invasive infection.

New Doppler index for prediction of perinatal brain damage in growth-restricted and hypoxic fetuses.

OBJECTIVE: To evaluate the new vascular score, hypoxia index (HI), in the prediction of sonographically detected structural brain lesions in neonates within the first week after delivery of growth-restricted fetuses. METHODS: This prospective study included 29 growth-restricted fetuses delivered between 31 and 40 gestational weeks. Doppler umbilical artery (UA) and middle cerebral artery (MCA) resistance indices (RI) were recorded at 48-h intervals for at least 2 weeks before delivery. The cerebroumbilical ratio (C/U ratio = MCA-RI/UA-RI) and the HI (the sum of the daily reductions in C/U ratio, i.e. percentage below the cut-off value of 1, over the period of observation) were calculated. After delivery, neonatal outcome was evaluated according to obstetric parameters and ultrasound examinations of the brain. Doppler indices, C/U ratio and HI, as well as neonatal clinical and biochemical parameters, were tested as potential predictors of brain lesions using the C4.5 data-mining algorithm. RESULTS: Neonatal brain lesions were detected in 13 growth-restricted fetuses. Of all the parameters tested by the C4.5 data-mining algorithm, only HI was identified as a predictor of neonatal brain lesions. HI also showed better correlation with neonatal biochemical parameters, such as umbilical venous partial pressure of oxygen and umbilical venous pH, compared with the C/U ratio. CONCLUSIONS: HI, which takes into account cumulative oxygen deficit, could significantly improve the prediction of a poor neurological outcome in pregnancies complicated by growth restriction and hypoxia.

Intraoperative dissemination of tumour cells in patients with Ewing tumours detected by RT-PCR.

Using reverse transcriptase polymerase chain reaction (RT-PCR) we evaluated the occurrence of tumour-cell ribonucleic acid (RNA) in the blood during surgery in patients with Ewing tumours. The patients received irradiation and chemotherapy according to the protocol of the European Intergroup Cooperative Ewing Sarcoma Study (EICESS) 92. Blood samples were taken from 15 patients. Intra-operative dissemination was found during 2/8 resections but showed no relation to patient survival. At second-look biopsy, detection of tumour-cell RNA was associated with relapse and metastases in 3/4 patients. The results suggest that pre-operative treatment did not completely prevent dissemination of tumour cells during surgery of Ewing tumours.

Modified fetal biophysical profile in the assessment of perinatal outcome.

The aim of the study is the evaluation of variables of the biophysical profile in the assessment of perinatal outcome. The prospective study included 87 pregnant women with singleton pregnancy in the 28th to 42nd week of gestation with clinically and ultrasonically verified fetal growth retardation, where the fetal biophysical profile was assessed antenatally. Through the factor analysis of biophysical profile variables we obtained values indicating the contribution of individual variables to the predictability of perinatal outcome. 70% of the patients were examined in 15 minutes according to the principles of modified biophysical profile. The most sensitive variable of the biophysical profile in the prediction of perinatal outcome was the amniotic fluid volume, followed by fetal breathing movements, non-stress test and fetal movements, while the lowest prediction value was assigned to the fetal tone. The modified biophysical profiles need to be perfected on a larger number of pregnant women, which would advance the predictability of this method in detection of hypoxically endangered fetuses.

Adenovirus E1A does not induce the Ewing tumor-associated gene fusion EWS-FLI1.

Rearrangement of the EWS gene with FLI1 is thought to occur early in the pathogenesis of Ewing's sarcoma family tumors (EFTs) because the chromosomal aberration is pathognomonic for this disease. Recently, adenovirus (Ad) 5 E1A protein has been reported to induce this gene rearrangement in a variety of cell types. This finding, if generally substantiated, not only suggests an etiological role for viral agents in the generation of oncogenic chromosomal aberrations but would also significantly impact the use of adenoviral vectors for gene therapy. In contrast, we now report on the absence of EWS-FLI1 chimeric products from short- and long-term cultures of stably Ad-transformed cells lines and from transiently E1A-expressing cell lines. In addition, we demonstrate the absence of E1A from EFTs. We conclude that there is no role for Ads in EFT pathogenesis. Consequently, evidence for a viral genesis of tumor-specific gene rearrangements is not available.

CD99 positivity and EWS-FLI1 gene rearrangement identify a breast tumor in a 60-year-old patient with attributes of the Ewing family of neoplasms.

Rearrangements of the EWS gene with ETS transcription factor genes as a result of chromosomal translocation and high expression levels of CD99MIC2 characterize the Ewing family of tumors (EFT). This group of rather undifferentiated neoplasms affects bone and soft tissue in children and young adults mostly between 5 and 30 years of age (median, 15 years). This study reports a case of a CD99MIC2 positive small round cell tumor in the breast of a 60-year-old woman in whom a t(11;22)(q24;q12) chromosomal aberration was identified by cytogenetic analysis. Reverse transcriptase (RT)-polymerase chain reaction (PCR) followed by sequence analysis revealed expression of a chimera transcript in which EWS exon 10 was fused to FLI1 exon 6. Previously, this gene fusion has been reported to occur in approximately 3% of EFT. The specific gene rearrangement of EWS intron 10 was confirmed on Southern blot of genomic DNA. This study further contributes to the growing list of unusual neoplasms in adults that carry genotypic and phenotypic traits of the EFT.

Cryptic exons as a source of increased diversity of Ewing tumor-associated EWS-FLI1 chimeric products.

In the Ewing family of tumors (EFT), the EWS gene is rearranged with members of the ets oncogene family. Variability in genomic breakpoint locations is the source of significant heterogeneity in fusion product structure. As a consequence of variably included exon sequences from the two partner genes, a variable amount of additional peptide sequence is inserted in between the minimal transforming domains. Some of this molecular diversity has recently been correlated with disparate clinical outcome. Here we report on cryptic exons found in the chimeric RNA of three EFT with different EWS-FLI1 fusions. In two tumors, the emergence of a cryptic exon from FLI1 intron 5 in the chimeric RNA was apparently unrelated to the genomic rearrangement that occurred in FLI1 introns 4 and 5, respectively. In one case, a novel exon was generated through the creation of an artificial splice acceptor site in FLI1 intron 6 by the genomic rearrangement that occurred in EWS intron 8. These results further extend the spectrum of molecular diversity in EFT.

Low incidence of molecular evidence for tumour in PBPC harvests from patients with high risk Ewing tumours.

Reverse transcriptase polymerase chain reaction (RT-PCR) was applied to evaluate the frequency of tumour cells in PBPC products from 15 high risk Ewing tumour (ET) patients who were treated according to EICESS 92 with high-dose chemotherapy (HDC) and stem cell rescue. Initial tumour cell contamination of the bone marrow (BM) detected by light microscopy was found in five and by RT-PCR in eight cases. RT-PCR was performed on each PBPC sample repeatedly at a sensitivity comparable to 20-100 highly EWS-Fli1 expressing tumour cells per 10 ml of fresh blood. Irrespective of the extent of BM involvement at diagnosis, all BM samples obtained before harvest were RT-PCR negative. Among 12 of 35 analysed apheresis products with single positive RT-PCR results only one sample tested reproducibly positive for tumour cell contamination in independent determinations. These preliminary data suggest that tumour cell contamination of PBPC is rarely found in patients with ET.

Predictive potential of testing for bone marrow involvement in Ewing tumor patients by RT-PCR: a preliminary evaluation.

EWS/ets-oncogene fusion transcripts can be detected in at least 98% of Ewing tumors [(ET) Ewing sarcoma and peripheral primitive neuroectodermal tumor] by reverse transcriptase-polymerase chain reaction (RT-PCR), thus confirming the histopathologic diagnosis. To detect minimal amounts of tumor cells in the bone marrow (BM), we used an RT-PCR assay with a high sensitivity, revealing one tumor cell in a background of 10(6) normal cells. We examined BM samples from 35 newly diagnosed ET patients (23 with localized and 12 with metastatic disease). At diagnosis, tumor cells in the BM were detected in 7/23 patients with localized disease (30%). Fifty percent of patients with isolated lung metastasis were RT-PCR positive (3/6), whereas 6/6 patients with bone metastases showed positive signals (100%). All patients with initial PCR positivity in the BM became negative during treatment. After a median follow-up of 30 months, relapses were observed in both groups of patients with localized disease (3/7 RT-PCR positive and 2/16 RT-PCR negative). The only recurrence in the group with isolated lung metastases occurred as progressive lung disease in 1 of the 2 RT-PCR-negative patients, whereas among the 6 patients with bone metastases 2 remain in complete remission. So far, RT-PCR screening for BM involvement did not allow prediction of early relapse in ET. To assess better the significance of this test in the evaluation of long-term prognosis and in monitoring the effectiveness of systemic therapy, long observation periods are warranted before it becomes a tool for treatment stratification.

Picornavirus 2A proteinase-mediated stimulation of internal initiation of translation is dependent on enzymatic activity and the cleavage products of cellular proteins.

Poliovirus and human rhinovirus 2A proteinases are known to stimulate translation initiation on the cognate viral Internal Ribosome Entry Segments (IRESes). The molecular mechanism of this translational transactivation was investigated in vitro using dicistronic mRNAs containing picornaviral IRESes as the intercistronic spacer and purified human rhinovirus type 2 and coxsackievirus B4 2A proteinases. The stimulation achieved on the HRV2 IRES in the presence of the cognate 2A proteinase at 1 microgram/ml was twofold; the maximum stimulation at 100 micrograms/ml was fivefold. The IRESes and proteinases from rhino- and enteroviruses were interchangeable; however, stimulation of translation initiation on a cardiovirus IRES by these proteinases was minimal. Studies using an inhibitor or a mutant 2A proteinase demonstrated that translation stimulation requires 2A-mediated enzymatic conversion of some cellular component(s). The HRV2 2A proteinase also stimulated translation initiation on full-length viral RNA, suggesting that 2A proteinase-mediated stimulation of IRES-driven translation has a physiological role.

Hemodynamic effects of mivacurium chloride administered to patients during oxygen-sufentanil anesthesia for coronary artery bypass grafting or valve replacement.

The hemodynamic effects of mivacurium chloride were studied in 54 adult cardiac patients anesthetized with midazolam and sufentanil. After baseline data were collected, a placebo (N = 9) or mivacurium was administered over 60 seconds, the latter in doses of 0.15 (N = 18), 0.20 (N = 18), or 0.25 (N = 9) mg/kg. Measurements were repeated 2, 5, and 10 minutes later. Baseline measurements were similar. A slight decrease in heart rate over time reached statistical significance in several groups including the control group. Mean arterial, mean pulmonary arterial, pulmonary arterial occlusion, and right atrial pressures and cardiac output did not change, nor did systemic and pulmonary vascular resistances and cardiac index. Besides the decrease in heart rate, the only hemodynamic change to reach statistical significance was an increase in stroke volume in patients given mivacurium 0.25 mg/kg. Significant hypotension occurred in two patients; in one, a sudden decrease in mean arterial pressure of 24% occurred 1 minute after mivacurium 0.20 mg/kg. Blood pressure was restored by ephedrine 10 mg. In the other patient, given mivacurium 0.25 mg/kg, mean arterial pressure decreased 50% from 73 to 37 mm Hg. Recovery was rapid without treatment. It is concluded that mivacurium administered in doses of 0.15 to 0.25 mg/kg over 60 seconds to cardiac patients is associated with few significant hemodynamic effects. However, a small number of patients may experience significant transient hypotension when given doses greater than of 0.15 mg/kg, two times the ED95.