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PubMed2000

Effects of amplification facilitators on diagnostic PCR in the presence of blood, feces, and meat.

The full potential of diagnostic PCR is limited, in part, by the presence of inhibitors in complex biological samples that reduce the amplification efficiency. Therefore, different pre-PCR treatments are being used to reduce the effects of PCR inhibitors. The aim of the present study was to investigate the effects of 16 amplification facilitators to enhance DNA amplification in the presence of blood, feces, or meat. Different concentrations of amplification facilitators and inhibitory samples were added to PCR mixtures containing rTth or Taq DNA polymerase. The addition of 0.6% (wt/vol) bovine serum albumin to reaction mixtures containing Taq DNA polymerase reduced the inhibitory effect of blood and allowed DNA amplification in the presence of 2% instead of 0.2% (vol/vol) blood. Furthermore, the addition of bovine serum albumin (BSA) to reaction mixtures containing feces or meat enhanced the amplification capacities of both polymerases. Taq DNA polymerase was able to amplify DNA in the presence of 4% instead of 0.4% (vol/vol) feces and 4% instead of 0.2% (vol/vol) meat, and rTth was able to amplify DNA in the presence of 4% instead of 0.4% (vol/vol) feces and 20% instead of 2% (vol/vol) meat. The single-stranded DNA binding T4 gene 32 protein (gp32) had a relieving effect similar to that of BSA, except when it was added to PCR mixtures of rTth containing meat and of Taq DNA polymerase containing feces. The relieving effects of betaine and a cocktail of proteinase inhibitors were more sample specific. The addition of 11.7% (wt/vol) betaine allowed Taq DNA polymerase to amplify DNA in the presence of 2% (vol/vol) blood, while the addition of proteinase inhibitors allowed DNA amplification by both polymerases in the presence of 4% (vol/vol) feces. When various combinations of betaine, BSA, gp32, and proteinase inhibitors were tested, no synergistic or additive effects were observed. The effects of facilitators on real-time DNA synthesis instead of conventional PCR were also studied.

Blood↗

PubMed2026

A small viral protein suppresses immune amplification by two distinct mechanisms.

Diverse viral suppressors of RNA interference (RNAi) and RNA silencing (VSRs) interact directly with core protein and/or RNA components of the host RNAi pathway. However, the specific counter-defense function of any VSR biochemical activity is fully validated only when it is shown as essential for viral infection in the wild-type but not mutant hosts defective in antiviral RNAi. Here, we investigated the role of VSR activities for direct binding to small-interfering RNA duplexes (siRNA), long double-stranded RNA (dsRNA), or RNA-dependent RNA polymerase 1 (RDR1) during plant infection by wild-type and mutant cucumber mosaic virus (CMV), a positive-strand RNA virus expressing the 110-residue 2b protein as its VSR. We demonstrate that a C-terminally truncated 2b mutant (2b1-93) active in direct binding to siRNA and dsRNA, but not RDR1, was able to suppress the amplification of virus-derived siRNAs (vsiRNA) and antiviral RNAi mediated by RDR6, but not RDR1. By contrast, an N-terminally truncated 2b mutant (2b18-110) inactive in direct binding to siRNA or dsRNA was able to suppress vsiRNA amplification and antiviral RNAi mediated by RDR1, but not RDR6, and was less effective to promote systemic CMV infection and disease development than 2b1-93. Together, our results show that whereas RDR1 suppression requires direct binding of VSR-2b to RDR1, but not siRNA or dsRNA, RDR6 suppression depends on direct binding to siRNA and dsRNA, but not RDR1. Therefore, CMV, through its VSR-2b, suppresses two parallel vsiRNA amplification pathways by distinct molecular mechanisms, and this unique property may account for the unusually wide host range of CMV.IMPORTANCEHost amplification of antiviral immunity is essential for robust control of viral infections. However, little is known about the mechanisms that viruses have evolved to suppress immune amplification in plants. Here, we characterized whole plant infection by cucumber mosaic virus (CMV) with its viral suppressor of RNA interference (RNAi) mutated to become inactive in direct binding to small-interfering RNA duplexes (siRNA), long double-stranded RNA (dsRNA), or RNA-dependent RNA polymerase 1 (RDR1). We demonstrate maximal suppression of both RDR1- and RDR6-mediated antiviral RNAi amplification by the CMV 2b protein, a viral suppressor of RNAi (VSR). Notably, whereas RDR1 suppression requires direct binding of 2b to RDR1 but not siRNA or dsRNA, RDR6 suppression depends on direct binding to siRNA and dsRNA, but not RDR1. Our findings reveal a novel counter-defense strategy evolved by a wide host range positive-strand RNA virus to suppress two pathways of immune amplification by distinct mechanisms.

Cucumovirus↗

PubMed2006

Amplification of the HER2 gene in breast cancers testing 2+ weak positive by HercepTest immunohistochemistry: false-positive or false-negative immunohistochemistry?

BACKGROUND: The majority of cases of breast cancer scoring HER2 weak positive (2+) on immunohistochemistry (IHC) using the HercepTest are not associated with amplification of the HER2/neu gene. AIM: To examine the reproducibility of IHC in cases scoring 2+ subsequently shown to have gene amplification by fluorescence in-situ hybridisation (FISH). METHODS: A retrospective analysis of 153 cases referred for FISH confirmation of a weak positive HercepTest (2+) result was performed. Repeat IHC was undertaken in cases with weak positive (2+) referral IHC and amplification of the HER2 gene by FISH. RESULTS: Amplification of the HER2 gene was confirmed in 29/153 cases (19%) scoring 2+ on IHC. Repeat IHC was carried out on 25 IHC 2+ cases: 7 (28%) scored 2+ on repeat IHC, 18 (72%) scored 3+ and were reclassified as strong positive. A heterogeneous expression pattern was present in 3/17 cases scoring 3+. CONCLUSIONS: The majority of HercepTest 2+ results are not accompanied by gene amplification and represent "false positive" IHC in terms of prognostic or therapeutic relevance. A small proportion of HercepTest 2+ scores represent true 2+ IHC positive cases accompanied by gene amplification: a category probably biologically related to 3+ IHC cases. The remainder of cases of HercepTest 2+ accompanied by gene amplification represent a category of referral IHC 2+ weak positive, FISH amplified, repeat IHC 3+ strong positive, best described as "false negative 2+ IHC". This has implications for selection of cases for FISH analysis where weak positive (2+) IHC score is used as a triage for FISH testing, and for testing strategies in referral laboratories undertaking FISH analysis.

Biomarkers, Tumor↗

PubMed2006

Fast amplification of dynamic synaptic inputs in spinal motoneurons in vivo.

The ability of voltage-dependent inward currents (likely Na(+)) of the adult cat lumbar motoneurons to amplify rapidly changing (i.e., dynamic) synaptic inputs was investigated using in vivo intracellular recording techniques. Fast amplification was assessed by measuring the magnitude of the high-frequency (180 Hz) component of the Ia synaptic input due to tendon vibration as a function of somatic voltage and was compared with the previously observed amplification of steady inputs (steady state response of PICs to slow inputs). Data from 17 experiments show that amplification of the dynamic input indeed occurred and was directly linked to neuromodulatory drive (standard state: decerebrate with intact descending neuromodulatory systems vs. minimal state: pentobarbital with said systems significantly inhibited). Fast amplification factors averaged 2.0 +/- 0.7 (mean +/- SD) in the standard neuromodulatory state. That is, the effective synaptic current was nearly twice as large at its peak as it was at hyperpolarized levels, ranging as high as 2.6. Although fast amplification was often smaller than the amplification of steady inputs, the difference was not statistically significant. However, the voltage at which fast amplification began was approximately 10 mV more depolarized (P < 0.01). It is concluded that both dynamic and steady inputs can be amplified, but there may be differences in mechanism.

Achilles Tendon↗

PubMed2001

CGH, cDNA and tissue microarray analyses implicate FGFR2 amplification in a small subset of breast tumors.

Multiple regions of the genome are often amplified during breast cancer development and progression, as evidenced in a number of published studies by comparative genomic hybridization (CGH). However, only relatively few target genes for such amplifications have been identified. Here, we indicate how small-scale commercially available cDNA and CGH microarray formats combined with the tissue microarray technology enable rapid identification of putative amplification target genes as well as analysis of their clinical significance. According to CGH, the SUM-52 breast cancer cell line harbors several high-level DNA amplification sites, including the 10q26 chromosomal region where the fibroblast growth factor receptor 2 (FGFR2) gene has been localized. High level amplification of FGFR2 in SUM-52 was identified using CGH analysis on a microarray of BAC clones. A cDNA microarray survey of 588 genes showed >40-fold overexpression of FGFR2. Finally, a tissue microarray based FISH analysis of 750 uncultured primary breast cancers demonstrated in vivo amplification of the FGFR2 gene in about 1% of the tumors. In conclusion, three consecutive microarray (CGH, cDNA and tissue) experiments revealed high-level amplification and overexpression of the FGFR2 in a breast cancer cell line, but only a low frequency of involvement in primary breast tumors. Applied to a genomic scale with larger arrays, this strategy should facilitate identification of the most important target genes for cytogenetic rearrangements, such as DNA amplification sites detected by conventional CGH. Figures on http://www.esacp.org/acp/2001/22-4/heiskanen.htm

Breast Neoplasms↗

PubMed2004

Induction of centrosome amplification and chromosome instability in human bladder cancer cells by p53 mutation and cyclin E overexpression.

Centrosome amplification frequently occurs in human cancers and is a major cause of chromosome instability (CIN). In mouse cells, centrosome amplification can be readily induced by loss or mutational inactivation of p53. In human cells, however, silencing of endogenous p53 alone does not induce centrosome amplification or CIN, although high degrees of correlation between p53 mutation and CIN/centrosome amplification in human cancer can be detected, suggesting the presence of additional regulatory mechanism(s) in human cells that ensures the numeral integrity of centrosomes and genomic integrity. Cyclin E, a regulatory subunit for CDK2 that plays a key role in centrosome duplication, frequently is overexpressed in human cancers. We found that cyclin E overexpression, together with loss of p53, efficiently induces centrosome amplification and CIN in human bladder cancer cells but not by either cyclin E overexpression or loss of p53 alone. We extended these findings to bladder cancer specimens and found that centrosome amplification is strongly correlated with concomitant occurrence of cyclin E overexpression and p53 inactivation but not with either cyclin E overexpression or p53 inactivation alone. Because cyclin E expression is strictly controlled in human cells compared with mouse cells, our findings suggest that this stringent regulation of cyclin E expression plays an additional role underlying numeral homeostasis of centrosomes in human cells and that deregulation of cyclin E expression, together with inactivation of p53, results in centrosome amplification.

Aged↗

PubMedDate not supplied

Does somatosensory amplification decrease with antidepressant treatment?

Somatosensory amplification refers to a tendency to experience somatic and visceral sensations as unusually intense, noxious, and disturbing. The authors wanted to determine whether somatosensory amplification is a stable construct or whether it might change with antidepressant therapy. Fifteen patients with fibromyalgia and 17 patients with major depressive disorder received antidepressant treatment and were assessed after 6 and 12 weeks of treatment. Amplification scores responded to antidepressant treatment in patients with major depression but not in patients with fibromyalgia, despite a decrease in the levels of depression in both groups. When change in depression and anxiety scores was partialled out from change in somatosensory amplification scores, the amplification scores did not change significantly in either the depressed or the fibromyalgia groups. Given the small numbers and the marginal significance of the results, the authors are unable to say definitely just how independent of depression somatosensory amplification is. Whether somatosensory amplification is a measure of depression per se should be tested in a more definitive and larger future study.

Adult↗

PubMed1996

A strategy for immunohistochemical signal enhancement by end-product amplification.

We report a novel strategy, called end-product (EP) amplification, capable of enhancing the sensitivity of immunohistochemical procedures by about an order of magnitude or more. The strategy employs an antibody (anti-EP) to the product generated by the action of horseradish peroxidase on 3,3'-diaminobenzidine (DAB), and can be extended to the products of other enzymes as well, e.g., alkaline phosphatase. Amplification is the consequence of the ability of anti-EP to detect the multiplicity of product moelcules resulting from the turnover of substrate by a single enzyme molecule. The subsequent detection of anti-EP was by biotinylated goat anti-rabbit antibody, followed by avidin-peroxidase and DAB or by avidin-alkaline phosphatase and Vector Red. Further amplification can be accomplished by repeated cycles of the protocol. Anti-EP was produced by immunization with a bovine serum albumin (BSA) conjugate of a soluble polymer of DAB, prepared by a carefully controlled reaction of DAB with horseradish peroxidase and hydrogen peroxide. Coupling to BSA (and to RSA) was accomplished with glutaraldehyde. The titer of anti-EP was established by ELISA. Formalin-fixed, paraffin-embedded sections of five cases of Hodgkin's disease and five tonsils with follicular hyperplasia were immunolabeled for the following lymphoid markers: CD3, CD20, CD30, CD45RA, and CD68. EP amplification with anti-EP was also applied to cases of CMV pneumonia and cerebral toxoplasmosis to determine whether this procedure could improve detection of the infectious agents. Immunolabeling of the primary antibody was performed by the avidin-biotin-peroxidase technique with DAB as the reaction substrate. The specificity of EP amplification was tested by demonstrating binding of anti-EP with Vector Red with the generation of a fluorescence end-point. There was complete congruence in the distribution of the DAB signal and the red immunofluorescence representing EP amplification. The intensity of the DAB signal was increased as much as 16-fold by EP amplification, making possible a reduction in the amount of the primary antibody by as much as 85-90%. Sensitivity also increased with respect to weakly expressed antigens and low concentrations of infectious agents.

3,3'-Diaminobenzidine↗

PubMed2005

Clinical implication of centrosome amplification in plasma cell neoplasm.

The mechanisms underlying aneuploidy in multiple myeloma (MM) are unclear. Centrosome amplification has been implicated as the cause of chromosomal instability in a variety of tumors and is a potential mechanism causing aneuploidy in MM. Using immunofluorescent (IF) staining, centrosome amplification was detected in 67% of monoclonal gammopathies, including monoclonal gammopathy of undetermined significance (MGUS). We also investigated the gene expression of centrosome proteins. Overall, gene expression data correlated well with IF-detected centrosome amplification, allowing us to derive a gene expression-based centrosome index (CI) as a surrogate for centrosome amplification. Clinically, MM patients with high CI (> 4) are associated with poor prognostic genetic and clinical subtypes (chromosome 13 deletion, t(4; 14), t(14;16), and PCLI > 1%, P < .05) and are shown here to have short survival (11.1 months versus 39.1 months, P < .001). On multivariate regression, a high CI is an independent prognostic factor. Given that centrosome amplification is already observed in MGUS and probably integral to early chromosomal instability and myeloma genesis, and patients with more extensive centrosome amplification have shorter survival, the mechanisms leading to centrosome amplification should be investigated because these may offer new avenues for therapeutic intervention.

Adult↗

PubMed1988

Selective amplification of luteinizing hormone by adenosine in rat luteal cells.

Adenosine amplification of LH-stimulated cAMP accumulation in rat luteal cells is rapid and dependent on mitochondrial ATP production. The objective of the present studies was to determine if this effect of adenosine is specific for LH and to gain information on the mechanism of the ATP-dependent amplification of LH action in rat luteal cells. Adenosine significantly amplified maximum cAMP accumulation in response to LH, isoproterenol, forskolin, and cholera toxin. However, amplification of this response by adenosine was significantly greater for LH than for the other agonists. The relative order of amplification by adenosine was LH greater than isoproterenol greater than forskolin greater than cholera toxin; the relative magnitudes of amplification by adenosine were 1, 0.6, 0.2, and 0.2, respectively. Neither LH, isoproterenol, forskolin, nor cholera toxin had any effect on cellular levels of ATP, and adenosine produced a similar rate of increase and maximal levels of ATP in the presence of all agonists. Ionomycin, a calcium ionophore, inhibited LH- and cholera toxin-stimulated cAMP accumulation and produced a dose-dependent depletion of ATP. Adenosine reversed the inhibitory effect of ionomycin on LH-stimulated cAMP accumulation and cellular levels of ATP. However, adenosine did not reverse the inhibitory effect of ionomycin on cholera toxin-stimulated cAMP accumulation, although its effects on cellular ATP levels were identical to those on LH. Thus, the selective amplification of LH by adenosine is not merely a substrate effect on adenylate cyclase activity. The nature of adenylate cyclase activation by cholera toxin and forskolin and the weak amplification by adenosine of these agonists compared to that of LH indicate that the site of the ATP-dependent action of adenosine appears to be before or on the G-protein of adenylate cyclase. We suggest that adenosine, by an ATP-dependent process, either increases the availability of functional LH receptors or increases coupling between the LH receptor and adenylate cyclase.

Adenosine↗

PubMed2001

Novel signal amplification technology with applications in DNA and protein detection systems.

A non-enzymatic approach to signal amplification has practical advantages over conventional target amplification methods. We have designed a simple, cost-efficient signal amplification system that can be used to enhance the detection of nucleic acids or protein. The signal amplification process requires initial capture of analyte by a specific probe, which, depending on the analyte, can be an oligomer or an antibody. Once the analyte is captured, amplification moieties are applied to significantly enhance the sensitivity of analyte detection. Nucleic acid amplification is typically greater than 1000-fold, increasing the sensitivity of target detection to less than 1 amol/100 microL. This amplification strategy presents a very flexible system with components that are easily altered to accommodate diverse assay requirements.

Animals↗

PubMed1998

Subcycling-PCR for multiplex long-distance amplification of regions with high and low GC content: application to the inversion hotspot in the factor VIII gene.

Previously we described a PCR protocol for detecting the inversion in the factor VIII gene, which is a common cause of Hemophilia A. This PCR assay is challenging due to the size of the amplification (10-12 kb), the varying GC content (30%-80%) and the multiplex PCR products involved (four for carrier female). Efficient amplification of the four segments depends on three unusual modifications to standard long-distance PCR protocols: (i) very high concentrations of dimethyl sulfoxide, (ii) addition of deaza-dGTP and (iii) high concentration of Taq and Pwo DNA polymerases. One of the segments was amplified much more efficiently than the others under standard three-temperature cycling conditions (12 s at 94 degrees C, 30 s at 65 degrees C and 14 min at 68 degrees C). To facilitate the uniform amplification of the multiple regions, subcycling-PCR (S-PCR) was developed. In S-PCR, the combined annealing/elongation step is composed of subcycles of shuttling between a low and a high temperature, e.g., shuttling four times between 60 degrees and 65 degrees C. S-PCR produces consistent robust amplification of the various segments produced by wild-type, mutant and carrier individuals. S-PCR is a simple generalization of PCR, which generally may be advantageous in three contexts: (i) amplification of long segments in which the GC content varies within the segment, (ii) multiplex amplification of long segments and (iii) multiplex amplification of short segments in which the GC content varies among the segments.

Chromosome Inversion↗

PubMed1999

Quantitative ratio of primer pairs and annealing temperature affecting PCR products in duplex amplification.

The quantity of PCR products that are simultaneously amplified from two different loci in a duplex amplification (DA) are significantly lower for one of the loci, as compared to identical PCR amplification in separate single-band amplifications (SBA). This difference in amplification probably occurs already after the second cycle of amplification. To further analyze this phenomenon, we tested different reaction conditions, including annealing times, a wide range of temperatures, various quantities of the template, several nucleotide concentrations, different amounts of TaqI DNA Polymerase, number of amplification cycles and various amounts of primers and primers ratio. Changing the ratio between the sets of primers in DA had the most significant effect on the relative levels of amplification of the loci with an optimal ratio of 4:1 in favor of the set of primers used to amplify the underrepresented fragment. The optimal annealing temperatures for the tested sets of primers were identical in SBA and different in DA. Possible reasons for this phenomenon are discussed.

Chromosomes, Human, Pair 1↗

PubMed2001

Technical note: A quick and more sensitive method to identify pork in processed and unprocessed food by PCR amplification of a new specific DNA fragment.

We developed and evaluated a PCR procedure to detect pork in heated and unheated meat, sausages, canned food, cured products, and pâtés using a faster, more specific, and more sensitive method than others previously described. Isolation of a new DNA-specific porcine repetitive element was performed by nonspecific PCR amplification. After analyzing this repetitive sequence, a pair of primers were synthesized. To confirm the effectiveness and specificity of this fragment, 55 pig blood DNA samples (from differents breeds) were tested and positive results were obtained. With 200 samples tested from other species, the specific pork amplification was not detected. Using this method, we can partially quantify degree of contamination, depending on the PCR amplification cycles, detecting up to 0.005% pork in beef and 1% pork in duck pâté using 30 and 20 PCR amplification cycles, respectively. The amount of porcine DNA detected in cattle DNA was 1.25 and 250 pg when using 30 and 20 amplification cycles, respectively. Pork has been identified in both heated and unheated meat products, sausages, canned food, hamburgers, and pâtés. In conclusion, specific PCR amplification of a repetitive DNA element seems to be a powerful technique for the identification of pork in processed and unprocessed food, because of its simplicity, specificity, and sensitivity (with 30 amplification cycles we can detect 0.005% pork). Furthermore, it is a very fast method, because 1% pork contamination can be detected with 20 PCR cycles. The procedure is also much cheaper than other methods based on RFLP-PCR, immunodiffusion, or other techniques that need expensive equipment.

Animals↗

PubMed1995

Consonant perception with linear and compression amplification.

The consonant perception of 15 subjects with mild to moderate sensorineural hearing loss was evaluated using linear amplification and two different types of compression amplification. A specially modified hearing aid was used which allowed for variation of the amplifier input/output function in three steps, such that the compression ratio could be set to 1 (linear), 1.3 or 1.8. The Nonsense Syllable Test (NST) was recorded through the aid in quiet and in two different noise conditions (four-talker babble and a background noise with sharp intermittent sounds), and replayed to the listeners through headphones. No differences in consonant perception were found between the different types of amplification in the quiet condition. In the babble condition, consonant perception was significantly better with linear amplification than with either form of compression. In the sharp noise condition, there was no difference in performance between linear amplification and compression amplification with the ratio of 1.8. Consonant perception was adversely affected, however, by the compression and amplification with the ratio of 1.3 in this condition. Overall NST results and results for particular classes of consonants are discussed.

Adult↗

PubMed2000

Adjustable amplification of synaptic input in the dendrites of spinal motoneurons in vivo.

The impact of neuromodulators on active dendritic conductances was investigated by the use of intracellular recording techniques in spinal motoneurons in the adult cat. The well known lack of voltage control of dendritic regions during voltage clamp applied at the soma was used to estimate dendritic amplification of a steady monosynaptic input generated by muscle spindle Ia afferents. In preparations deeply anesthetized with pentobarbital, Ia current either decreased with depolarization or underwent a modest increase at membrane potentials above -40 mV. In unanesthetized decerebrate preparations (which have tonic activity in axons originating in the brainstem and releasing serotonin or norepinephrine), active dendritic currents caused strong amplification of Ia input. In the range of -50 to -40 mV, peak Ia current was over four times as large as that in the pentobarbital-anesthetized preparations. Exogenous administration of a noradrenergic agonist in addition to the tonic activity further enhanced amplification (sixfold increase). Amplification was not seen in preparations with spinal transections. Overall, the dendritic amplification with moderate or strong neuromodulatory drive was estimated to be large enough to allow the motoneurons innervating slow muscle fibers to be driven to their maximum force levels by remarkably small synaptic inputs. In these cells, the main role of synaptic input may be to control the activation of a highly excitable dendritic tree. The neuromodulatory control of synaptic amplification provides motor commands with the potential to adjust the level of amplification to suit the demands of different motor tasks.

Afferent Pathways↗

PubMed2005

Detection of Her2/neu, c-MYC and ZNF217 gene amplification during breast cancer progression using fluorescence in situ hybridization.

The deletion of tumor suppressor genes and amplification of activating oncogenes appear to be critical events in tumorigenesis. We carried out fluorescence in situ hybridization (FISH) analysis of the breast cancer cell line MCF7 and clinically obtained cancer tissue sections on the basis of which we suggest a breast cancer development model. We selected 28 genes for FISH probes from breast cancer patients. Of the 28 genes studied, 14 were shown to be consistently amplified in the breast cancer cell line MCF7. We selected three genes from clinical tumor samples on the basis of results from MCF7 analysis. The amplification of Her2/neu or ZNF217 is closely associated with the stages of breast cancer. The frequency of amplification of Her2/neu increased notably in patients at stages later than IIB based on TNM staging, whereas the amplification of ZNF217 correlated with T2N1M0 at stage IIB and later stages. c-MYC amplification was not related to the stage. Her2/neu, ZNF217 and c-MYC were found to have a significantly increased copy number in breast cancer cells. In breast cancer progression, c-MYC amplification is an early event, while ZNF217 and Her2/neu amplification may play a role in the later stage of tumor development.

Breast Neoplasms↗

PubMed1997

Malignant astrocytoma-derived region of common amplification in chromosomal band 17p12 is frequently amplified in high-grade osteosarcomas.

Recently, we reported a new amplification event that involves marker D17S67 in 17p12 in three malignant astrocytomas of patients with a very short survival. The amplified region may contain an oncogene implicated in astrocytoma tumorigenesis. To determine the extent of the amplified regions, we constructed a yeast artificial chromosome contig spanning the D17S67 region and tested the amplification status of markers that map to the contig. We determined a commonly amplified region between markers D17S1311 and D17S1875 with a maximal length of 1,630 kb. By using marker 745R, from within the commonly amplified region, we screened 60 high-grade astrocytomas but could not detect additional tumors with the amplification event. This suggests that the incidence of the amplification event in high-grade astrocytoma is low (5%). It has recently been shown by comparative genomic hybridization that amplification of 17p11-p12 is a frequent event in high-grade osteosarcomas, occurring in 20-30% of cases. Since the commonly amplified region is within 17p12, we tested 745R in 20 osteosarcomas, including 6 lung metastases, and detected amplification in 9 cases (45%). Marker 745R was found to be amplified in 4 of the 6 lung metastases (66%). From this frequent involvement and the association with clinically aggressive astrocytomas we conclude that for both tumor types presence of the amplification event seems to correlate with aggressive clinical behaviour.

Astrocytoma↗