Increased differentiation and production of extracellular matrix components of primary human osteoblasts after cocultivation with endothelial cells: A quantitative proteomics approach.

Coculturing of bone-forming and blood vessel-forming cells is a strategy aimed at increasing vascularity of implanted bone constructs in tissue-engineering applications. We previously described that the coculture of primary human osteoblasts (hOBs) and human umbilical vein endothelial cells (HUVECs) improves the differentiation of both cell types, leading to the formation of functional blood vessels and enhanced bone regeneration. The objective of this study was to further delineate the multifaceted interactions between both cell types. To investigate the proteome of hOBs after cocultivation with HUVECs we used stable isotope labeling by amino acids in cell culture, revealing 49 significantly upregulated, and 54 significantly downregulated proteins. Amongst the highest regulated proteins, we found the proteins important for osteoblast differentiation, cellular adhesion, and extracellular matrix function, notably: connective tissue growth factor, desmoplakin, galectin-3, and cyclin-dependent kinase 6. The findings were confirmed by enzyme-linked immunosorbent assays. We also investigated whether the mRNA transcripts correlate with the changes in protein levels by quantitative real-time reverse transcription polymerase chain reaction. In addition, the data was compared to our previous microarray analysis of hOB transcriptome. Taken together, this in-depth analysis delivers reliable data suggesting the importance of coculturing of hOBs and HUVECs in tissue engineering.

The adipofasciocutaneous gluteal fold perforator flap a versatile alternative choice for covering perineal defects.

AIM: Perineal defects following the resection of anorectal malignancies are a reconstructive challenge. Flaps based on the rectus abdominis muscle have several drawbacks. Regional perforator flaps may be a suitable alternative. We present our experience of using the gluteal fold flap (GFF) for reconstructing perineal and pelvic defects. METHODS: We used a retrospective chart review and follow-up examinations focusing on epidemiological, oncological (procedure and outcome), and therapy-related data. This included postoperative complications and their management, length of hospital stay, and time to heal. RESULTS: Twenty-two GFFs (unilateral n = 8; bilateral n = 7) were performed in 15 patients (nine women and six men; anal squamous cell carcinoma n = 8; rectal adenocarcinoma n = 7; mean age 65.5 + 8.2 years) with a mean follow-up time of 1 year. Of the cases, 73.3% were a recurrent disease. Microscopic tumor resection was achieved in all but one case (93.3%). Seven cases had no complications (46.7%). Surgical complications were classified according to the Clavien-Dindo system (grades I n = 2; II n = 2; IIIb n = 4). These were mainly wound healing disorders that did not affect mobilization or discharge. The time to discharge was 22 + 9.9 days. The oncological outcomes were as follows: 53.3% of the patients had no evidence of disease, 20% had metastatic disease, 20% had local recurrent disease, and one patient (6.7%) died of other causes. CONCLUSIONS: The GFF is a robust, reliable flap suitable for perineal and pelvic reconstruction. It can be raised quickly and easily, has an acceptable complication rate and donor site morbidity, and does not affect the abdominal wall.

Randomized in situ study on the efficacy of CO2 laser irradiation in increasing enamel erosion resistance.

OBJECTIVES: The aim of this double-blind, randomized in situ study was to evaluate the erosion-preventive effect of a specific set of CO2 laser parameters, associated or not with fluoride. METHODS: Two hundred forty bovine enamel blocks were prepared for individual palatal appliances (n = 6 samples/appliance). The study had four phases of 5 days each, with ten volunteers and the following treatments: CO2 laser irradiation (L), fluoride treatment (F), combined fluoride and laser treatment (FL), and no treatment, control (C). Laser irradiation was performed at 0.3 J/cm2 (5 µs/226 Hz/10.6 µm) and the fluoride gel contained AmF/NaF (12'500 ppm F-/pH = 4.8-6). For erosive demineralization, the appliances were immersed extra-orally in citric acid (0.05 M/20 min/pH = 2.3) twice daily. Analysis of enamel surface loss was done using a 3D-laser profilometer on 3 days. Additionally, fluoride uptake was quantified and scanning electron microscopies were done. Data were analyzed with repeated measures ANOVA and post hoc pairwise comparisons (α = 0.05). RESULTS: At all analyzing days, both laser groups caused the lowest means of enamel loss, which were also statistically significant lower than C (p < 0.05). At day 5, FL means ± SD (33.6 ± 12.6 µm) were even significantly lower than all other groups (C 67.8 ± 15.4 µm; F 57.5 ± 20.3 µm; L 46.8 ± 14.5 µm). Significantly increased enamel fluoride uptake was observed for both fluoride-containing groups (p < 0.05) at day 1. CONCLUSION: Compared to the control, the CO2 laser irradiation with a specific set of laser parameters (0.3 J/cm2/5 µs/226 Hz) either alone or in combination with a fluoride gel (AmF/NaF) could significantly decrease enamel erosive loss up to 5 days in situ. CLINICAL RELEVANCE: Combined CO2 laser-fluoride treatment has a significant anti-erosive effect.

Overexpression of Hif-1α in Mesenchymal Stem Cells Affects Cell-Autonomous Angiogenic and Osteogenic Parameters.

Reconstruction of large bone defects still represents a major medical challenge. In recent years tissue engineering has developed techniques based on adult mesenchymal stem cells (MSCs) that could represent an attractive therapeutical option to treat large bone defects in the future. It has been demonstrated in various animal models that ex vivo expanded MSCs are capable of promoting the regeneration of skeletal defects after implantation. However, for the efficient regeneration of bone in tissue engineering applications, a rapid vascularization of implanted grafts is essential to ensure the survival of cells in the early post-implantational phase. A promising strategy to enhance vascularization of MSC-containing implants could consist of overexpression of the angiogenic master transcription factor Hypoxia-inducible factor 1 (Hif-1) in the MSCs in order to induce angiogenesis and support osteogenesis. In the present study, we overexpressed Hif-1α in MSCs by using recombinant adenoviruses and investigated cell-autonomous effects. Overexpression of Hif-1α enhanced proliferation, migration, cell survival and expression of pro-angiogenic genes. Other parameters such as expression of the osteogenic markers BMP-2 and RunX2 were decreased. Hif-1α overexpression had no effect on invasion, senescence and osteogenic differentiation of MSCs. Our experiments revealed multifarious effects of Hif-1α overexpression on cell-autonomous parameters. Therefore, Hif-1α overexpression may represent a therapeutic option to improve cellular functions of MSCs to treat critical sized bone defects.

Increased extracellular matrix and proangiogenic factor transcription in endothelial cells after cocultivation with primary human osteoblasts.

The most promising strategies in bone engineering have concentrated on providing sufficient vascularization to support the newly forming tissue. In this context, recent research in the field has focused on studying the complex interactions between bone forming and endothelial cells. Our previous work has demonstrated that direct contact cocultivation of human umbilical vein endothelial cells (HUVECs) with primary human osteoblasts (hOBs) induces the osteogenic phenotype and survival of hOBs. In order to investigate the mechanisms that lead to this effect, we performed microarray gene expression profiling on HUVECs following cocultivation with hOBs. Our data reveal profound transcriptomic changes that are dependent on direct cell contact between these cell populations. Pathway analysis using the MetaCore™ platform and literature research suggested a striking upregulation of transcripts related to extracellular matrix and cell-matrix interactions. Upregulation of a number of major angiogenetic factors confirms previous observations that HUVECs enter a proangiogenic state upon cocultivation with osteoblasts. Interestingly, the downregulated transcripts clustered predominantly around cell cycle-related processes. The microarray data were confirmed by quantitative real-time RT-PCR on selected genes. Taken together, this study provides a platform for further inquiries in complex interactions between endothelial cells and osteoblasts.

Cytogenetic aspects of childhood leukemias.

Recurrent non-random chromosome abnormalities, including numerical or structural changes such as translocations, inversions, insertions or deletions within the leukemia cell nucleus, have been discovered in approximately 80% of patients with a malignant hematological disease. These reciprocal translocations correlate with specific cellular subtypes of hematopoeisis at the stage of their maturation arrest and are therefore important for diagnosis. Some of these aberrations are independent prognostic indicators and help to stratify patients into different risk-adapted therapy groups. Owing to new laboratory methods such as the fluorescence in situ hybridization (FISH) and modified polymerase chain reaction (RT-PCR) the chromosomal breakpoints can be investigated and the rearrangements of genes which produce the abnormal proteins can be identified. Due to the high sensitivity of these available data a new prognostic factor, the "minimal residual disease" (MRD), can be investigated at diagnosis and at intervals during the treatment period. Since we now know which oncoproteins are involved, a target-directed therapy with inhibitors might be possible in the future.Standard cytogenetic and molecular genetic analysis of the leukemia karyotype is of the utmost importance for classification (WHO), therapy and therapy results in the acute childhood leukemias.

Denervation of the thumb carpometacarpal joint for symptomatic osteoarthritis in 46 patients.

We investigated the effect of an extended denervation procedure in the thumb carpometacarpal (CMC) joint in patients suffering from CMC osteoarthritis. Between 2006 and 2018, 46 patients underwent the procedure in our clinic and were included in this retrospective study. Pain, strength, range of motion, DASH score, complications and overall satisfaction were determined. Assessment showed a significant decrease in pain and excellent physical function at a median 5 years' follow-up. Twelve patients needed secondary surgery due to persistent pain. Overall, 28 of the 46 patients were satisfied with the results of the denervation. Even though the results of CMC denervation are poorer than with simple trapeziectomy, considerable pain relief can be achieved in selected young, physically active patients by exclusively soft-tissue surgery, conserving the biomechanical properties of the joint. In case of failure of the procedure, all other options remain available.

Herpes simplex virus: dry mass.

Dry mass of herpes simplex virus particles was measured by quantitative electron microscopy after isolation by surface spreading and critical-point drying of infected cells. The core weighed about 2 x 10(-16) gram, the empty naked capsid 5 x 10(-16) gram, the full naked capsid 7 x 10(-16) gram, and the enveloped nucleocapsid 13 x 10(-16) gram.

CO2 Laser (10.6 microm) parameters for caries prevention in dental enamel.

Although CO(2) laser irradiation can decrease enamel demineralisation, it has still not been clarified which laser wavelength and which irradiation conditions represent the optimum parameters for application as preventive treatment. The aim of the present explorative study was to find low-fluence CO(2) laser (lambda = 10.6 microm) parameters resulting in a maximum caries-preventive effect with the least thermal damage. Different laser parameters were systematically evaluated in 3 steps. In the first experiment, 5 fluences of 0.1, 0.3, 0.4, 0.5 and 0.6 J/cm(2), combined with high repetition rates and 10 micros pulse duration, were chosen for the experiments. In a second experiment, the influence of different pulse durations (5, 10, 20, 30 and 50 micros) on the demineralisation of dental enamel was assessed. Finally, 3 different irradiation times (2, 5 and 9 s) were tested in a third experiment. In total, 276 bovine enamel blocks were used for the experiments. An 8-day pH-cycling regime was performed after the laser treatment. Demineralisation was assessed by lesion depth measurements with a polarised light microscope, and morphological changes were assessed with a scanning electron microscope. Irradiation with 0.3 J/cm(2), 5 micros, 226 Hz for 9 s (2,036 overlapping pulses) increased caries resistance by up to 81% compared to the control and was even significantly better than fluoride application (25%, p < 0.0001). Scanning electron microscopy examination did not reveal any obvious damage caused by the laser irradiation.

[Comparison of the Results of Lundborg's and Sirotakova's Resection-Suspension Arthroplasty for the Treatment of Trapeziometacarpal Joint Osteoarthritis]. / Vergleich der Ergebnisse der Resektions-Suspensionsarthroplastik nach Lundborg und Sirotakova zur Behandlung der Rhizarthrose.

BACKGROUND AND AIMS: The aim of this prospective randomised study was to compare Sirotakova's and Lundborg's methods of resection-suspension arthroplasty using the abductor pollicis longus tendon in the surgical treatment of osteoarthritis of the trapeziometacarpal joint. PATIENTS AND METHODS: Between 2009 and 2012, 38 patients (29 female, 9 male) with symptomatic trapeziometacarpal osteoarthritis (34% stage II, 58% stage III and 8% stage IV according to the Eaton-Littler classification) were randomly allocated to one of the surgical methods (mean age 62.7 years, range 43-85). Preoperatively, the following data were collected: pain intensity (Visual Analogue Scale, VAS; at rest: Lundborg 4.4±1.7; Sirotakova 4.6±2.1), strength (key pinch force Lundborg 8.1 kPa±6.6; Sirotakova 10.4 kPa±10.8), range of motion in the trapeziometacarpal joint (Lundborg 61.64±26.4; Sirotakova 46.67±25.6), Kapandji index (Lundborg 9.42±1.4; Sirotakova 9.33±1.5), distance between the base of the first metacarpal bone and the scaphoid bone as measured by standardised x-ray images (Lundborg 12 mm±1.5; Sirotakova 11.4 mm±3), DASH questionnaire (Lundborg 40.4±13.9; Sirotakova 49.9±23.5). A significant difference between the 2 groups was not found. Patients were examined 3 and 9 months postoperatively. RESULTS: Both resection-suspension arthroplasty procedures led to a statistically significant postoperative reduction of pain, a significant improvement in radial and palmar abduction, a significant gain in quality of life and significant asymptomatic proximalisation of the first metacarpal bone. There was no significant difference in postoperative strength. CONCLUSION: Both methods lead to reliable and satisfying results. Given our findings we cannot generally recommend one method over the other.

[Current Perceptions of Lipofilling on the Basis of the New Guideline on "Autologous Fat Grafting"]. / Aktuelle Erkenntnisse zur Eigenfett Transplantation anhand der neuen Leitlinie "Autologe Fetttransplantation".

Introduction: Autologous fat transfer has recently become an increasingly popular surgical procedure and comprises harvesting, processing and transplantation of adipose tissue, as well as professional follow-up care. This method, as a surgical procedure, can be utilised for trauma-, disease- or age-related soft tissue volume deficits and soft tissue augmentation. As usage is increasing, but the variables of fat harvest, specific indications and fashion of fat transfer are poorly defined, there is a great demand for development of a guideline in the field of reconstructive and aesthetic surgery. Methods: All relevant points were discussed within the scope of a consensus conference including a nominal group process of all societies involved in the procedure and ratified with a strong consensus (>95%). Literature from the standard medical databases over the last 10 years was retrieved, studied and specific guidelines were concluded. Results: Consensus was achieved among all professionals involved on the following points: 1. definition 2. indication/contraindication, 3. preoperative measures 4. donor sites 5. techniques of processing 6. transplantation 7. follow-up care 8. storage 9. efficacy 10. documentation 11. evaluation of patient safety. Conclusion: Definite indications and professional expertise are paramount for autologous fat tissue transfer. Successful transfers are based on the use of correct methods as well as specific instruments and materials. Autologous adipose tissue transplantation is considered to be a safe procedure in reconstructive and aesthetic surgery, due to the low rate of postoperative complications and sequelae.

Neuroblastoma: inverse relationship between expression of N-myc and NGF-r.

12 primary neuroblastomas (NB) of different maturation stages, 2 ganglioneuromas (GN), and 2 neuroblastoma cell lines were analysed for RNA expression of the protooncogene N-myc and the gene encoding the nerve growth factor receptor (NGF-r) by Northern-blots, RNA-dot-blots and for receptor presence by immunohistological procedures. In 4 tumors with strongly elevated RNA expression of N-myc the NGF-r RNA expression was weak or absent. In all 8 tumors with highly increased NGF-r transcription no N-myc expression was detectable. These results, indicating an inverse relationship between N-myc and NGF-r expression, could help in establishing markers for differentiation, and thus prognosis, in neuroblastoma.

The T-ALL specific t(11;14)(p13;q11) translocation breakpoint cluster region is located near to the Wilms' tumour predisposition locus.

A breakpoint cluster region (T-ALLbcr) has been previously described on 11p13 for T-ALL carrying t(11;14)(p13;q11). One further T-ALL breakpoint is described bringing to 5 out of 6 such translocations which are found to break within a maximum of 6.7 kb on chromosome 11p13. Studies of somatic cell hybrids derived from t(11;14)(p13;q11) T-ALL placed the T-ALLbcr between the genes for catalase (CAT) and the beta-subunit of follicle stimulating hormone (FSHB). This suggested a link between the T-ALLbcr and the Wilms' tumour predisposition locus (WT) since constitutional 11p13 deletions predispose to Wilms' tumour. Utilising somatic cell hybrids from patients with Wilms' tumours and aniridia, we show that while the T-ALLbcr maps distal to the catalase gene at 11p13, it maps outside the shortest region of overlap of a series of 11p13 deletions associated with Wilms'-Aniridia. The data suggest the order of genes at 11p13 to be: centromere-CAT-T-ALLbcr-WT-aniridia-FSHB-telomere. Therefore, the T-ALLbcr must lie very close to but may be distinct from the Wilms' predisposition locus at 11p13.

Cloning and characterization of AFX, the gene that fuses to MLL in acute leukemias with a t(X;11)(q13;q23).

We report the cloning and characterization of the entire AFX gene which fuses to MLL in acute leukemias with a t(X;ll)(q13;q23). AFX consists of two exons and encodes for a protein of 501 amino acids. We found that normal B- and T-cells contain similar levels of AFX mRNA and that both the MLL/AFX as well as the AFX/MLL fusion transcripts are present in the cell line and the ANLL sample with a t(X;11)(q13;q23). The single intron of the AFX gene consists of 3706 nucleotides. It contains five simple sequence repeats with lengths of at least 12 bps, a chi-like octamer sequence (GCA/TGGA/TGG) and several immunoglobulin heptamer-like sequences (GATAGTG) that are distributed throughout the entire AFX intron sequence. In the KARPAS 45 cell line the breakpoints occur at nucleotides 2913/2914 of the AFX intron and at nucleotides 4900/4901 of the breakpoint cluster region of the MLL gene. The AFX protein belongs to the forkhead protein family. It is highly homologous to the human FKHR protein, the gene of which is disrupted by the t(2;13)(q35;q14), a chromosome rearrangement characteristic of alveolar rhabdomyosarcomas. It is noteworthy that the t(X;11)(q13;q23) in the KARPAS 45 cell line and in one acute nonlymphoblastic leukemia (ANLL) disrupts the forkhead domain of the AFX protein exactly at the same amino acids as does the t(2;13)(q35;q14) in case of the FKHR protein. In addition, the 5'-part of the AFX protein contains a conserved hexapeptide motif (QIYEWM) that is homologous to the functionally important conserved hexapeptide QIYPWM upstream of the homeobox domain in Hox proteins. This motif mediates the co-operative DNA binding of Pbx family members and Hox proteins and, therefore, plays an important role in physiologic and oncogenic processes. In acute leukemias with a t(X;11)(q13;q23), this hexapeptide motif is separated from the remaining forkhead domain within the AFX protein. The predicted amino acid sequence of AFX differs significantly from the partial AFX protein sequence published previously (Genes, Chromosomes and Cancer, 1994, 11, 79-84). This discrepancy can be explained by the occurrence of two sequencing errors in the earlier work at nucleotide number 783 and 844 (loss of a cytosine residue or guanosine residue, respectively) that lead to two reading frame shifts.

A DNA damage repair mechanism is involved in the origin of chromosomal translocations t(4;11) in primary leukemic cells.

Some chromosomal translocations involved in the origin of leukemias and lymphomas are due to malfunctions of the recombinatorial machinery of immunoglobulin and T-cell receptor-genes. This mechanism has also been proposed for translocations t(4;11)(q21;q23), which are regularly associated with acute pro-B cell leukemias in early childhood. Here, reciprocal chromosomal breakpoints in primary biopsy material of fourteen t(4;11)-leukemia patients were analysed. In all cases, duplications, deletions and inversions of less than a few hundred nucleotides indicative of malfunctioning DNA repair mechanisms were observed. We concluded that these translocation events were initiated by several DNA strand breaks on both participating chromosomes and subsequent DNA repair by 'error-prone-repair' mechanisms, but not by the action of recombinases of the immune system.

Applied Sciences: Absence of CD44-standard in human neuroblastoma correlates with histological dedifferentiation, N-myc amplification and reduced survival probability.

Expression of CD44 was examined by immunohistochemistry in 205 primary neuroblastomas together with histological grading according to the Shimada classification at the time of diagnosis. In addition, Southern blot analysis to determine N-myc gene amplification was carried out in the same tissue. When compared with clinical data such as stage, age and event-free survival probability it was found that CD44 expression characterizes well differentiated tumours and thus correlates with prognosis (event-free survival probability in CD44 positive patients 0.6 (n = 129) vs. 0.0 (n = 21) in CD44 negative patients). In tumours with N-myc = 1, CD44 positivity was found in 91% of patients as compared to 57% of patients with N-myc > 1 in tumours. All tumours of 13 patients with metastatic stage 4s (with good prognosis) showed CD44s expression. Thus, detection of CD44s expression might serve as a prognostic indicator which can be rapidly detected at diagnosis.

A new fingerprint method for sequence analysis of chromosomal translocations at the genomic DNA level.

Chromosomal rearrangements constitute a significant feature of leukemogenesis and malignant transformation in general. Nucleotide patterns in the immediate vicinity of the break point may provide important information about the underlying causalities, eg illegitimate recombination events mediated by topoisomerase II, Alu repeats, or VDJ recombinase. In order to facilitate the determination of those DNA patterns, we developed a new fingerprint approach. In a first step, two DNA fragments were independently amplified by long distance PCR: the genomic region carrying the break point and the normal nonrearranged counterpart. Subsequently, both PCR products were digested with restriction enzymes, end-labelled with a fluorescent dye, and subjected to high resolution polyacrylamide gel electrophoresis. By comparing the restriction patterns of the rearranged and the nonrearranged PCR fragments, the break points could be easily localized within a size range coverable by a single sequencing reaction. Finally, the exact DNA sequence across the break point was directly determined. The 'fingerprint' technique is fast, reliable and enables the assay of multiple samples in parallel.

Molecular analysis of MLL-1/AF4 recombination in infant acute lymphoblastic leukemia.

We examined ten cases of acute lymphoblastic leukemia (ALL) in infants (less than 1 year of age) by RT-nested PCR for a MLL-1/AF4 rearrangement. Five patients revealed a positive result. The specific PCR product differed in size from approximately 380-670 bp indicating various splicing variants in the MLL-1/AF4 rearrangement. Three patients had a fusion between exon 6 of the MLL-1 gene and codon 362 of the known AF4 cDNA sequence. Moreover, in two patients more than one specific PCR product was detected, possibly due to alternative splicing. In the first case, sequencing of these products revealed a hybrid mRNA consisting of MLI-1 exon 7 or exon 8, respectively, fused to the AF4 gene at codon 348. In the second case with alternative splicing, again, exon 7 or 8 of the MLL-1 gene were fused to the AF4 gene as in case 1. The AF4 sequence involved in this patient, however, started at codon 362. The AF4 break was, therefore, identical to the three MLL-1/AF4 positive patients as described above. Moreover, we investigated all ten patients for the reciprocal mRNA transcript AF4/MLL-1 by a similar PCR approach. In none of these patients, including the five MLL-1/AF4 positive cases was a specific PCR product obtained. However, in the MV411 cell line bearing a t(4;11), which served as a positive control in our MLL-1/AF4-PCR assay, the reciprocal AF4/MLL-1 mRNA was detected. Our results indicate that a MLL-1/AF4 rearrangement occurs in about 50% of infants with ALL. In contrast, the reciprocal hybrid mRNA can only rarely be detected, if at all.

Detection of hyperdiploid karyotypes (>50 chromosomes) in childhood acute lymphoblastic leukemia (ALL) using fluorescence in situ hybridization (FISH).

ALL patients with a hyperdiploid karyotype of more than 50 chromosomes (high hyperdiploidy) carry a better prognosis in contrast to patients presenting with other cytogenetic features, and an appropriate less intensive therapy protocol should be developed for these patients. For this reason it is desirable to have a quick screening method identifying those with this type of hyperdiploidy. We therefore studied the bone marrow and/or blood cells of 278 children with ALL using double target fluorescence in situ hybridization (FISH) on interphase. A combination of DNA probes (repetitive, centromere specific) was applied detecting chromosomes which are most frequently overrepresented in patients with hyperdiploidy (>50), at chromosomes 6, 10, 17 and 18. All patients showing hybridization signals differing from the normal signal distribution of two spots for each tested chromosome were analyzed cytogenetically as well. 102 children (102/278; 36.7%) were found to have a clone with aberrant FISH results. In 80 patients (80/278, 28.8%) the cytogenetic analysis detected a hyperdiploid karyotype >50 chromosomes, whereas the remaining patients (n=12) could be related to other ploidy subgroups, ie hyperdiploidy with 47-50 chromosomes, haploidy, triploidy/tetraploidy. Comparison of the FISH results with the measurements of the DNA content showed good agreement for 88.8% (208/234) of the investigated patients. The detected rate of 28.8% patients with a high hyperdiploid karyotype in our investigated cohort is comparable to the frequency of other studies. Only one patient was not identified as having a hyperdiploid karyotype with our combination of DNA probes. Our results indicate that FISH is a feasible and quick screening method for the detection of hyperdiploid karyotypes (>50 chromosomes) and other ploidy subgroups.

Rapid synthesis of hybrid RNA molecules associated with leukemia-specific chromosomal translocations.

A number of gene arrangements have been described as characteristic abnormalities associated with different types of leukemia, and this list is still growing. In view of the biological, clinical and prognostic relevance of the pathological fusion products, techniques permitting their detection are of paramount importance in the clinical setting. In some instances, permanent leukemic cell lines carrying the abnormality of interest are available for the establishment and standardization of molecular assays. For a number of newly discovered gene rearrangements, however, this may not be the case. It is therefore of great interest for clinical laboratories to have alternative technical possibilities for the set-up of standardized molecular tests. This problem provided the stimulus to design a simple and rapid method for in vitro generation of chimeric RNA molecules corresponding to pathological fusion transcripts typical for chromosomal translocations in leukemias. Two separate fragments are generated in a four-primer multiplex PCR. Due to a PCR-generated overlap, a chimeric fragment can be synthesized in a second round of PCR. This PCR product is then purified with the help of magnetic beads. Due to the SP6 promotor sequence incorporated during the second round of PCR, transcription into RNA is easily facilitated while the template DNA is still bound to the solid phase. Following this strategy we were able to synthesize the fusion transcripts m-BCR/ABL, CBF beta/MYH11, and MLL/AFp1 which are the molecular equivalents of t(9;22)(q34,q11), inv16(p13;q22) and t(1;11)(p32;q23), respectively. The chimeric RNA will be useful as a control template in diagnostic RT-PCR strategies. It can also be further processed in translation systems leading to the corresponding chimeric oncoprotein. This approach can be easily used to create any hybrid RNA of interest.