Biomedical subjects
Publications and source records attributed to C Mannhalter.
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It would be important to estimate in advance the risk of recurrent thrombosis. Deficiencies of antithrombin, protein C or protein S, or resistance to activated protein C are associated with a biochemically detectable prethrombotic state. It is thus far unknown whether in patients with a history of thromboembolism but without a defined clotting abnormality a heightened coagulation activation is detectable. We investigated the value of prothrombin fragment F1+2 (F1+2) as a predictor of recurrent venous thromboembolism. Furthermore, we compared the F1+2 levels of thrombosis patients without a defined clotting defect to those of Factor V Leiden patients with a history of venous thrombosis and to those of healthy controls. 180 patients without a defined clotting abnormality and 73 patients with Factor V Leiden were prospectively followed after discontinuation of oral anticoagulants for venous thrombosis and F1+2 was measured at regular intervals. Recurrent venous thromboembolism occurred in 23 (9%) of the 253 patients. Before or at several time points after oral anticoagulants, no significant difference in F1+2 levels was found in patients with and without recurrent thrombosis. F1+2 levels at 3 weeks and prior to recurrence were not significantly different in both patient groups. Over a one-year observation period, F1+2 levels of both patients with and without Factor V Leiden were higher than those of the controls. No difference in F1+2 was seen between patients with and without Factor V Leiden. We conclude that monitoring of F1+2 is not suitable for identification of individuals at risk of recurrent venous thrombosis. Permanent hemostatic system activation is detectable both in patients with a defined abnormality of the clotting system and in patients in whom a particular defect has not (yet) been identified.
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Between February 1982 and April 1995, 62 patients (37 male, 25 female) with acute myelogenous leukemia (AML) with a median age of 32 years (19-51 years) received allogeneic marrow grafts from an HLA-identical sibling (n=60) or an HLA-mismatched family member (n=2). At the time of transplant, 35 patients were in first complete remission (CR), five in second CR, eight were primary refractory, eight were in untreated relapse and six in refractory relapse. The FAB subtypes were as follows: M1 (n=17), M2 (n=13), M3 (n=6), M4 (n=19), M5 (n=6), M6 (n=1). For conditioning most patients were given total body irradiation combined with cyclophosphamide (CY, n=50) or CY and busulfan (n=9). For graft-versus-host disease prophylaxis patients received cyclosporin A (CSA) and methotrexate (MTX) (n=32), MTX alone (n=12), CSA and methylprednisone (n=5), or CSA alone (n=13). As of April 1995, probability of leukemia-free survival projected at 10 years after BMT was 60% for patients transplanted in first CR compared with 10% for patients transplanted beyond first CR. Transplant-related mortality was 11% after BMT in first CR and 39% after BMT beyond first CR. Probability of relapse projected at 10 years after BMT is 32% for patients who received transplants in first CR and 81% for patients who received transplants beyond first CR. Thus, high-dose chemo/radiotherapy followed by allogeneic marrow infusion has a high curative potential for patients with AML who receive transplants in first CR and offers the chance of long-term disease-free survival for some patients with advanced disease.
A heterogeneous group of patients suffering from adenomatous polyposis coli (APC) were evaluated by clinical and genetic investigations for the first time in Austria. The patients belonged to eight unrelated APC families. In six families several family members were affected with APC, and linkage analysis with highly informative markers was used to estimate the risk of single individuals in these families to develop APC. All index patients were also tested for the most frequent mutation in the APC gene (mutation cluster region, exon 15). Clinical investigations included ophthalmologic tests for congenital hypertrophy of retinal pigment epithelium and colonoscopy. According to DNA analysis, 5 of 19 at-risk individuals had to be considered to be at high risk of having inherited the disease. Four of them underwent proctocolectomy, one patient at risk is under colonoscopic surveillance. The predictive value of indirect genotype analyses reached 83.3%; direct mutation analyses allowed risk estimation in 50% of cases. Ophthalmologic investigation was informative in 75% of the families. Direct and indirect genotyping using a panel of highly polymorphic, closely linked microsatellite markers is a valuable, rapid, reliable method for establishing a presymptomatic diagnosis of APC, especially in families in which more than one affected individual is available for analysis. With regard to the onset of APC and extracolonic manifestations, the variability of APC demands clinical investigations in addition to the molecular tests for all patients and their first-degree relatives.
Twenty-two patients with refractory or relapsed AML were treated with FLAG [25 mg/m2 fludarabine daily (days 1-5), 2 g/m2 daily Ara-C (days 1-5) and 400 micrograms/m2 daily G-CSF (day -1 till the absolute neutrophil count was > 500/microliter)]. Median age was 46 years (range 24-63). Eight patients had leukemia which was primarily refractory to conventional regimens, six were in first, seven were in second, and one was in third relapse. Overall, 11 of 22 (50%) patients achieved complete remission (CR), three had a partial response (PR), and seven did not respond (NR). One patient died of an early cerebral hemorrhage. The median remission duration from achievement of CR after FLAG was 9.9 months and median survival was 13.0 months. One patient is alive in CR at 31.9 months. Hematological toxicity of the regimen was severe. The median time to neutrophil recovery (ANC > 500/microliter) was 21 days (range 18-33). A median of seven red cell units (range 0-22) and of six platelet concentrate units (range 3-28) had to be given. Median duration of febrile neutropenia was 2 days (range 0-20 days) and patients were on i.v. antibiotics for a median of 16 days (range 0-51). There was no death from infection. Nonhematological toxicity was remarkably low, with almost no neurotoxicity and no major hepatotoxicity. In conclusion, FLAG seems to be an efficient and well tolerated regimen. It may be particularly useful for patients who have a sibling or unrelated donor for subsequent allogeneic bone marrow transplantation.
A number of different polymorphisms have been observed in coding as well as in non-coding regions of T cell receptor (TCR) genes. We report the identification and characterization of a highly polymorphic locus in the 3' noncoding region of the human T cell receptor alpha/delta (TCRAD) on chromosome 14. In 202 unrelated individuals, ten different alleles were distinguished by polymerase chain reaction (PCR) and a heterozygosity rate of 64% was calculated. Sequence analysis revealed that this polymorphic region consists of 10 bp imperfect repeat units and represents a variable number tandem repeat region (VNTR). Stable Mendelian inheritance of this novel polymorphic marker was proven in four families. The localization of this VNTR polymorphism in the TCRAD locus should make it a useful system for linkage analysis in immunological disorders with a known role of TCRAD.
When solitary pulmonary tumors are observed in patients with a history of cancer, differentiation between metastasis and primary lung cancer is crucial for appropriate therapy. Assuming that p53 mutations are conserved in metastases, mutation analysis of the p53 gene would be a valuable tool in differentiating metastases from primary carcinomas of the lung. In nine of 267 resected lung tumors, the origin of the lung tumor could not be defined histologically. Five patients had a history of colorectal carcinoma, one had a history of breast carcinoma, one had a history of soft-tissue carcinoma, and one had a history of head and neck carcinoma. One patient with a clear cell carcinoma of the lung had been surgically treated for both renal and thyroid cancer. Material from one patient with adenocarcinoma of the lung, histologically defined regional lymph nodes, and distant brain metastasis served as a control. We extracted deoxyribonucleic acid from the snap-frozen tissue of the unclassified lung tumors, from paraffin-embedded tissue of the previously removed primary cancers, and also from peripheral blood of the patients. Exons 2 to 11 of the p53 gene were amplified in separated polymerase chain reactions and directly sequenced. In all cases, the presence of germline mutations was excluded by analysis of peripheral blood deoxyribonucleic acid. The p53 mutation detected in the deoxyribonucleic acid of the lung tumor of the control patient proved to be conserved in the lymph nodes as well as in the brain metastasis. In two cases, the lung tumors exhibited a p53 mutation not present in the previously removed primary tumor and were therefore classified as new primary lung cancers. In five cases, the lung tumors proved to be metastases of the first tumor, exhibiting the identical p53 mutation. One of these lung tumor samples could be identified as a metastasis from the renal cancer, but the corresponding thyroid cancer material was different. For two cases, molecular analysis remained inconclusive. In one case, no p53 mutation could be found in the compared samples; in the other, no deoxyribonucleic acid could be extracted. Analysis of p53 mutations allowed exact classification in tumors for which standard methods failed to distinguish between metastasis or primary tumor. More than two thirds of lung tumors in patients with previous gastrointestinal carcinoma were revealed to be metastases, but second primary lung cancer could also be diagnosed. This diagnosis allowed correct surgical and adjuvant treatment of these patients.
In 29 patients (17 females) homozygous Arg 506 Gln mutation (FV Leiden) was identified. 25 had been investigated because of venous thromboembolism (VTE); four asymptomatic patients were found during family studies. The first VTE had occurred significantly earlier in females (median age [m] 26 years, range 17-49) than in males (m=38 years, range 21-82) (P=0.01). 12 females (80%) had taken oral contraceptives (OC, estrogen content 0.02-0.1 mg) for 6-150 months prior to thrombosis. Further triggering conditions in females were hormone replacement (n=1) and pregnancy (n=2). In 8/10 males the first VTE had occurred spontaneously--in two after surgery. The sites of VTE were deep vein thrombosis, pulmonary embolism, caval vein thrombosis and superficial thrombophlebitis. From our data we conclude that OC medication is the most important precipitating factor for VTE in females with homozygous FV Leiden.
We describe a multiplex polymerase chain reaction (PCR) method suitable for the detection of all T-cell receptor (TCR) gamma-chain gene rearrangements in patients with lymphoproliferative diseases. 40 patients with various lymphoproliferative disorders and 40 healthy individuals were tested. Clonal TCR gamma rearrangements were identified in all patients with malignant disorders, and in one of 10 cases with established reactive lymphocytosis but not in normal controls. In all individuals testing positive, the patient's specific V and J segment involved in the rearrangement could be determined by simply splitting the multiplex primer mix. Our data show that the multiplex PCR technique enables rapid, simple and sensitive screening for clonal TCR gamma chain gene rearrangements.
We report a patient with chronic myeloid leukaemia who underwent allogeneic marrow transplantation (BMT) but had a molecular relapse 5 months and haematological relapse 15 months after BMT. Since therapy with alpha-interferon had been ineffective he received leucocyte infusions from his sibling donor. He developed acute graft-versus-host disease and became aplastic 6 weeks later. Despite donor marrow infusion and cytokine stimulation marrow aplasia persisted for 13 weeks. Then, donors' peripheral blood stem cells were given after conditioning with cyclophosphamide and antithymocyte globulin resulting in trilineage engraftment of donor haemopoiesis. Since then, the patient has been in continuous molecular remission for 11 months.
Screening for a resistance against activated protein C (aPCR), which is in most cases caused by FV:Q506 mutation, is performed by functional tests measuring the effect of aPC on activated partial thromboplastin time (aPTT). Because of an insufficient discrimination between FV:Q506 mutation negative and positive individuals with the first generation of the functional test Coatest aPC Resistance (Chromogenix AB, Mölndal, Sweden), the definition of an arbitrary cut-off level was only possible using the results of DNA analysis. The use of an arbitrary cut-off level still resulted in unsatisfactory low sensitivity and specificity for the functional test. Thus, time- and cost-consuming DNA analyses had to be performed frequently to establish the diagnosis. The objective of this study was to evaluate an improved version of this assay that uses predilution of samples with factor V deficient plasma containing a heparin neutralizer. Using the data from 32 FV:Q506 mutation positive and 55 mutation negative individuals, the authors calculated a cut-off value resulting in an enhanced sensitivity (0.91 versus 1.0) and specificity (0.77 versus 1.0) compared to the old one. Imprecision was lowered from 5.36% (first generation) to 2.43%, in particular in samples with longer clotting times. In patients with prolonged aPTT, either caused by therapy with oral anticoagulants or heparin, correct results were obtained with the second generation assay, in contrast to the first generation assay. With this second generation assay the number of DNA analyses can be substantially reduced.
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BACKGROUND: Vascular access thrombosis represents a serious problem in haemodialysis patients. Therefore identification of relevant thrombotic risk factors is clinically valuable. Resistance to activated protein C (APC) was recently identified as a new thrombophilic defect which is caused by a single point mutation in the factor V gene. Whether this mutation predisposes to vascular access thrombosis is unknown. METHODS: The presence of factor V Leiden (mutation at nucleotide position 1691 of the factor V gene) was determined by polymerase chain reaction (PCR) analysis in 152 haemodialysis patients from all three haemodialysis units of the University Hospital of Vienna. In 61 patients (54 without mutation, 7 with heterozygous mutation) resistance to APC was evaluated. One hundred-seven individuals without renal failure (57 negative for factor V Leiden, 50 heterozygous subjects) served as controls. Haemodialysis patients with heterozygous factor V Leiden mutation were carefully investigated for thrombotic complications of vascular access, other thromboembolic events and additional putative thromboembolic risk factors. RESULTS: Seven of 152 (4.6%) patients were heterozygous carriers of factor V Leiden. The mean APC resistance ration in heterozygous dialysis patients was 2.31; in the 50 heterozygous controls the ratio was 2.02. The mean APC ratio in haemodialysis patients without mutation was 3.53 in contrast to 2.95 in the control group. Not one of the seven heterozygous haemodialysis patients suffered from vascular access thrombosis of inexplicable origin. Three patients remained totally free of access thrombosis from onset of haemodialysis treatment. In four of seven patients nine events of thrombosis of the vascular access occurred, but were due to anatomical stenosis in each case. In six permanent central venous catheters no episode of occlusion or reduced blood flow requiring thrombolytic therapy was observed. Family history with regard to thrombotic events was negative in all seven patients. No thromboembolic complication occurred during 13 periods of immobilization, in the course of six pregnancies and during oral contraception. CONCLUSIONS: The heterozygous carrier status for factor V Leiden does not appear to represent a risk factor for vascular access thrombosis in haemodialysis patients. This is possibly due to the fact that the functional APC activity is high and in heterozygous haemodialysis patients APC resistance ratios are very close to the normal range. However, it cannot be excluded that a homozygous factor V mutation represents an increased risk for shunt thrombosis. Therefore patients suffering from repeated and/or inexplicable shunt thrombosis should be tested for the factor V mutation to evaluate the effect of a homozygous mutation.
Activated protein C (APC) resistance is a common risk factor for venous thromboembolism and is associated with the replacement of Arg 506 by Gln in the factor V gene (factor V Leiden). We investigated the risk of recurrence of venous thromboembolism in APC resistant patients heterozygous for FV Leiden and compared these patient groups with a group of patients, who had a history of venous thromboembolism, but had neither APC resistance nor the FV Leiden mutation. APC resistance was determined in frozen blood samples from patients with a history of venous thromboembolism, who were not receiving oral anticoagulant (OAC) treatment. The plasma samples were collected between 1984 and 1991. Twenty-one patients in whom APC resistance was found in the stored plasma samples were reinvestigated in 1994 (5 males, 16 females, median [m] age 49 years, range 21-71 years). Twenty-one sex and age matched patients with venous thromboembolism (5 males, 16 females, age m = 50 years, range 25-73 years) investigated during the same time period who had a normal APC resistance test served as a control group. Patients with APC resistance as well as controls were reinvestigated for the presence of FV Leiden by genetic analysis in 1994. Of the 21 APC resistant patients, 5 were homozygous and 16 heterozygous for FV Leiden. Before the study entry homozygous patients had a significantly higher recurrence rate (5/5 patients) compared to the control group in heterozygous patients (9/16) and controls (9/21) the recurrence rate was not significantly different. The total observation time was 21 years in patients with homozygous FV Leiden, 83 years in patients with heterozygous FV Leiden and 108 years in controls, excluding the time when patients were on OAC treatment. During the observation time the recurrence rate was highest in patients with homozygous FV Leiden (9.5% per patient per year), but was similar in patients with heterozygous FV Leiden (4.8% per patient per year) and controls (5% per patient per year). Two of five (40%) homozygous patients, 4/16 (25%) heterozygous and 5/21 (24%) controls had a least one recurrent event during the observation period. The probability for development of thrombosis in the Kaplan-Meyer-Plot analysis was not different between the three groups. Bearing limitations of our study in mind (retrospective design, relatively small patient number) we conclude that the risk of recurrence after a thromboembolic event is not higher in patients with heterozygous FV Leiden than in patients without this mutation. Thus, it appears that the identification of heterozygous FV Leiden mutation is not an indication for long-term OAC treatment. Also, long-term OAC treatment cannot generally be recommended for homozygous patients with a single thromboembolic event. More definitive conclusions will require larger prospective studies.
An allogeneic sex-mismatched BMT which was performed in a male patient with BCR-ABL-positive ALL in second hematological and central nervous system relapse resulted in a CR for 12 months. After BMT, the patient was closely monitored with reverse transcription (RT)-PCR. One month before a third relapse RT-PCR became positive. During relapse G-CSF was administered. It specifically stimulated the donor-derived myelopoiesis and led to the stabilization of the disease for 8 months. Fluorescence in situ hybridization analyses of individual cell populations revealed that during the whole course of G-CSF administration granulocytes, CD4+, CD8+ and CD34+/CD10- cells were of female (donor) origin and only the CD34+/CD10+ cells which represented the leukemic blasts, were of male (host) origin.
Two new polymorphisms were identified in the protein S gene (PROS1): an intronic T/A-dimorphism (PIPS1) in intron K, and an exonic C/A-dimorphism (PEPS2), located in the 3'untranslated trailer of exon 15. Allelic frequencies of 24% (PIPS1-A) and 17% (PEPS2-A) respectively, were determined in the normal population. The identification of an intronic and an exonic PROS1 dimorphism, in addition to the known BstXI dimorphism, enlarges the molecular tool box for gene analysis and transcript quantification in hereditary protein S deficiency. Haplotype analysis showed that variability of both new polymorphisms occurred almost exclusively in the A-allele of the known intragenic BstXI dimorphism. Therefore, PEPS2 and PIPS1 are especially valuable in individuals homozygous for the BstXI A-variant.