The use of spermHALO-FISH to determine DAZ gene copy number

The AZFc region of the human Y chromosome is frequently deleted in men with spermatogenic failure and contains many multicopy genes. The best-characterized gene family within this region is the Deleted in AZoospermia (DAZ) gene family, which is present in four nearly identical copies. Recent reports claim deletions of some but not all DAZ genes. The assays used in these studies, however, are unable to provide conclusive evidence on the number of DAZ genes. In this study we show that with the use of highly decondensed sperm nuclei with large DNA domains (spermHALO) it is possible to determine the number of DAZ genes accurately. Using this fluorescent in-situ hybridization (FISH) technique, which has both high resolution and high range, we show that in 10 normospermic men, in which PCR digest assays indicated a deletion of one or more DAZ genes, all four DAZ genes were present. Also we confirmed previous findings of a deletion of two DAZ genes in two men and identified a man with six DAZ genes. Our results indicate that spermHALO-FISH allows an accurate determination of DAZ gene copy number, while PCR digest assays do not. Therefore, confirmation of positive results from PCR digest assays with spermHALO-FISH is essential. Furthermore, the spermHALO-FISH technique should prove useful as a genetic mapping technique in other regions of the Y chromosome and similar repetitive regions throughout the genome.     

The status of HPV16-specific T-cell reactivity in health and disease as a guide to HPV vaccine development

Human papilloma viruses (HPV) are among the most common sexually transmitted pathogens in young adults. In the majority of individuals, anti-viral immunity is capable of suppressing viral infection but in a minority of patients viral infection is not cleared in time to prevent the development of malignancies. In these cases, HPV16-specific immunity may develop too late, is not strong enough, and/or is possibly of the wrong type. The influence of pre-existing immunity on the efficacy of vaccines is largely unknown. Nor has it been studied what the effect is of vaccines on the various types of pre-existing HPV-specific T-cell immunity. Animal models showing that vaccines are able to protect against a subsequent tumor challenge and even to treat transplantable tumors, are not qualified to address this point because tumor development is not preceded by persistent viral infection. Therefore, the comparison between fully characterized pre-existing HPV-specific immunity in patients and healthy subjects is a prerequisite for the full appreciation of vaccine-efficacy as well as for further development of next-generation vaccines.     

Sorption of thiotepa to polyurethane catheter causes falsely elevated plasma levels

Central venous access catheters are commonly used in clinical oncology. The double lumen variant is applied in pharmacokinetic studies for simultaneous administration and blood sampling when frequent blood collections are necessary. Occlusion of one lumen, a common complication, necessitates the investigator perform blood sampling through the administration lumen after interrupting the infusion. Plasma concentrations measured in this sample can be influenced by sorption of the previously infused compound to the catheter lumen. In this study, the quality of cyclophosphamide, thiotepa, and carboplatin plasma concentrations is investigated when sampling is performed through the administration lumen.     

Toxicity of the high-dose chemotherapy CTC regimen (cyclophosphamide,thiotepa, carboplatin): the Netherlands Cancer Institute experience

High-dose chemotherapy (HD-CT) has a role in the potentially curative treatment of several tumours. The relative efficacies of the different regimens have not been studied in comparative trials, but it is clear that toxicities differ significantly between them. We analysed the immediate and long-term toxicity in the first 100 consecutive patients treated with the CTC regimen (cyclophosphamide 6000 mg m(-2), carboplatin 1600 mg m(-2) (or 20 mg ml(-1) min under the curve (AUC)) both as daily 1 h infusion, thiotepa 480 mg m(-2) as twice daily 30 min infusion, all divided over 4 consecutive days) followed by peripheral blood progenitor cell reinfusion (PBPC-Tx). Most patients had high-risk (n=86) or metastatic (n=4) breast cancer, or a germ cell tumour (n=8). Two patients (with a medulloblastoma and an aesthesioneuroblastoma, respectively) received CTC as off-protocol salvage regimen. The main toxicity was bone marrow suppression. Most patients had PBPC-Tx with granulocyte colony-stimulating factor (G-CSF), and the median time to neutrophil count 500 x 10(6) l(-1) and platelet count >20 x 10(9) l(-1) without transfusion independence was 10 (range 8-25) and 13 (8-60) days, respectively. The toxic death rate was 1%. Other frequent toxicities were neutropenic fever requiring antibiotics (n=65), central catheter-related infection (n=12) or a bleeding episode (n=48), mostly epistaxis (n=26). Reversible cardiac toxicity was seen in six patients and pulmonary events occurred in seven patients (infection (n=6), embolism (n=1)). Grade 3-4 gastrointestinal toxicity was frequent: nausea and vomiting 55%, diarrhoea 28% and mild liver toxicity (transaminase elevations) 9%. One patient pretreated with cisplatin had a kidney transplantation 8 years after HD-CT. Late complications included reversible radiation pneumonitis (n=12) and chronic heart failure (n=2). We found five second solid malignancies and two myelodysplasias. In conclusion, the CTC regimen is associated with a moderate, mainly reversible, toxicity. Future studies need to compare the efficacy and toxicity of the different HD-CT regimens.     

Marker genes for circulating tumour cells predict survival in metastasized breast cancer patients

We investigated the prognostic significance of circulating breast cancer cells in peripheral blood detected by quantitative RT-PCR of marker genes in patients with advanced breast cancer. Blood samples from 94 breast cancer patients with metastatic disease (M1) were examined for circulating tumour cells by studying the mRNA expression of CK19, p1B, PS2 and EGP2 by real-time PCR. Using a score function, developed for predicting circulating tumour cells by quadratic discriminant analysis (QDA), the four expression levels were combined into a single discriminant value. Tumour cells were present in 24 out of 94 (31%) of the patients. In 77% (72 out of 94) of the patients distant metastatic disease was localised in the bone. In 36% (26 out of 72) of the patients with bone metastases at the time of blood sampling, a positive QDA for the four genes was found, in contrast to only 14% (three out of 22) without bone involvement. Overall survival rates by Kaplan-Meier revealed no prognostic effect for the presence of bone metastases (P=0.93). However, patients with a positive QDA value did have a progression-free survival at 1 year of 3% and overall survival at 2 years of 17%, against 22 and 36% for patients with a negative QDA value (P=0.015 and 0.0053, respectively). Breast cancer patients with metastatic disease have a significantly worse progression-free and overall survival when circulating tumour cells can be detected in their peripheral blood.     

Latent Cerebral Venous and Sinus Thrombosis

The incidence of cerebral venous and sinus thrombosis (CVST) is still unknown. Several assumptions of the incidence have been made. In the one and only series of consecutive brain autopsies series by Towbin in1973 CVST was found in 10,9 % of patients aged 60 years or more. These findings suggest that the true incidence of CVST is higher than generally thought, but there are no other reports supporting these findings. Therefore in order to determine the incidence of latent CVST we conducted a new prospective post mortem study. All brain autopsies performed in our hospital between 1996 and 1998 on patients 60 years of age or older were examined for the presence of CVST in a prospective way. We examined the sagittal sinus, the torcula, the straight sinus, both transverse sinuses, and both sigmoideal sinuses for the presence of thrombosis. The clinical condition, medication, brain-imaging results, clinical cause of death, general findings from the body autopsy and definite cause of death were recorded. Additionally we requested the annual mortality figures of CVST at the department of the National Agency for Statistics of the Netherlands (CBS). A consecutive and prospective series of 102 brain autopsies was performed in our general hospital during a period of two years. We found one case of latent CVST in our series of 102 patients. Comparison with the findings of Towbin using a chi-square test yielded a significant difference in the incidence of latent CVST (χ₂ = 7.65, p < 0.01) between the two studies. The present autopsy study demonstrates that latent CVST is rare. Received: 7 June 2002, Received in revised form: 14 October 2002, Accepted: 21 October 2002 Correspondence to H. P. Bienfait, MD     

The identification of trends in the utilisation of mental health services by elderly: a Dutch case register study

OBJECTIVE: In view of the rapid ageing of the population any changes in the use of mental health services by the elderly became increasingly important for policy development. This study aimed at the supply of information about trends in the numbers of elderly clients, the services they used and the volume and pattern of service utilisation. METHODS: Details of elderly users and their use of community- and hospital-based services between 1990 and 1999 were retrieved from the Groningen case register. Developments in population size and age distribution in the register area were taken into account, as were the unit costs of mental health services. RESULTS: Large age specific changes were found that caused only the expenditures on the oldest elderly to increase due to a shift from outpatient clinics to prolonged psychogeriatric day treatment and inpatient care. Comparatively young elderly used fewer inpatient services and more community care. The number of new elderly clients declined progressively. In some age groups treated prevalence also decreased, but to a lesser extent, because of a prolonged use of mental health services. CONCLUSIONS: Study results seemed well in accordance with mental health policy as to deinstitutionalization and active ageing. Research on the effect of mental health care on life expectancy and the time lag between the intake of mental health providers and treated prevalence was proposed in order to improve the prediction of future service use by elderly.     

Urinary excretion of thioTEPA and its metabolites in patients treated with high-dose cyclophosphamide, thioTEPA and carboplatin.

The urinary excretion of N,N',N"-triethylenethiophosphoramide (thioTEPA), and its metabolites N,N',N"-triethylenephosphoramide (TEPA), N,N'-diethylene,N"-2-chloroethylphosphoramide (monochloroTEPA) and thioTEPA--mercapturate was determined in patients receiving thioTEPA as part of a high-dose combination chemotherapy regimen with cyclophosphamide and carboplatin. The thioTEPA dose was 40 or 60 mg/m(2) in short infusions, twice daily, during 4 days. Urine samples were collected after each voiding on each day of drug administration until 24--48 h after the last thioTEPA infusion. ThioTEPA, TEPA and monochloroTEPA concentrations were determined with gas chromatography and thioTEPA--mercapturate with liquid chromatography--mass spectrometry with direct sample injection. ThioTEPA was present in urine 30 min after infusion and was still excreted 18 h after the last infusion. All metabolites were detected in urine 1 h after infusion. Patients with a creatinine clearance above 140 ml/minl showed higher excretion of TEPA than patients with a creatinine clearance below 140 ml/min (12.8 versus 4.9%, p=0.01). The excretion of monochloroTEPA relative to the excreted amount of TEPA increased at lower pH values of the urine. The excretion of thioTEPA--mercapturate relative to the dose was higher in patients treated with 60 mg/m(2). Excretion of thioTEPA and monochloroTEPA both accounted for only 0.5% of the dose, while TEPA and thioTEPA--mercapturate both accounted for 11.1%.     

Ultrastructural evidence of early non-fibrillar Aβ42 in the capillary basement membrane of patients with hereditary cerebral hemorrhage with amyloidosis, Dutch type

The C-terminal profile and ultrastructure of small and presumably early capillary amyloid β protein (Aβ) deposits were investigated in four patients with hereditary cerebral hemorrhage with amyloidosis, Dutch type. The C terminus of the 40 (Aβ40) or the 42 (Aβ42) amino acid form of Aβ was gold labeled in serial, ultrathin sections on glass slides for reflection contrast microscopy and on grids for electron microscopy. In all studied subjects, reflection contrast microscopy revealed capillaries with focal Aβ42 immunolabeling in the absence of Aβ40 labeling. In the adjacent electron microscopic section, Aβ42 labeling was confined to the capillary basement membrane. The majority of Aβ42⁺40^– deposits showed no amyloid fibrils. Aβ42⁺40^– deposits were sometimes observed in an unremarkable basement membrane but usually showed increased electron density and reticular structures. A small subset of Aβ42⁺40^– deposits with basement membrane changes showed few amyloid fibrils. Aβ42⁺40⁺ capillary deposits always showed definite fibrils and were larger than Aβ42⁺40^– capillary deposits. The present findings suggest that in capillaries the accumulation and subsequent polymerization of Aβ42, possibly in conjunction with basement membrane changes, precedes the definite fibril formation with Aβ40. Received: 9 June 1999 / Accepted: 9 September 1999     

Topoisomerase I/II inhibitor intoplicine administered as a 24 h infusion: phase I and pharmacologic study

Intoplicine, an antitumor drug which interacts with both topoisomerase enzymes I and II, has demonstrated a broad spectrum of activity in preclinical studies. This indicates further clinical evaluation. In the present phase I study, with the primary objective to determine the maximum tolerated dose, intoplicine was administered by a 24 h continuous infusion every 21 days to 32 patients with solid malignant tumors. The patients received 12-640 mg/m2 by a central venous catheter. Liver toxicity was dose limiting. One patient died in a hepatic coma after the first course (dose 640 mg/m2), which was associated with intoplicine treatment. Other side effects were sporadic and mild. Myelotoxicity was virtually absent. Twenty-two patients had stable disease for four to six courses of treatment. The plasma concentration-time curves were compatible with standard linear pharmacokinetic models, with a protracted terminal half-life (mean 115 h). Although one sudden death occurred probably due to intoplicine toxicity, we nevertheless feel that research with intoplicine should continue, mainly because of its preclinical activity and its unique mechanism of action. The recommended dose for phase II studies with intoplicine administered as a 24 h infusion is 384 mg/m2. Liver toxicity, also seen in studies employing other dosages and infusion durations, should be investigated extensively in further clinical studies.