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Results for “amplification”

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PubMed2003

[Loss of p53 gene and amplification of HER-2 oncogene in primary hepatocellular carcinoma and their clinical significance].

OBJECTIVE: To investigate the deletion of p53 gene and amplification of HER-2 oncogene at chromosome 17 in primary hepatocellular carcinoma (HCC) and the clinical significance. METHODS: Interphase dual fluorescence in situ hybridization (FISH) was applied to detect the ratio of the number of p53 gene copy or HER-2 oncogene copy to that of chromosome 17 copy, to determine the p53 gene deletion and HER-2 oncogene amplification in nuclei prepared from 42 surgical specimens of HCC. Statistical analysis for their clinical significance was performed. RESULTS: Loss of p53 gene and amplification of HER-2 oncogene were detected in 27 (64.3%) and 9 (21.4%) of the 42 HCC respectively including 4 cases with low and 5 with high copy amplification. Six (14.3%) of 42 HCC showed simultaneously p53 gene deletion and HER-2 oncogene amplification. 61.9% (26/42) of HCC were polysomy 17, which correlated positively with p53 gene deletion (chi(2) = 12.286, P < 0.001). No close correlation between p53 gene loss and HER-2 oncogene amplification was found (chi(2) = 0.00, P = 1.00). Loss of p53 gene was related to the serum alpha-fetoprotein (AFP) level and the tumor size (P < 0.05). The postoperative 2-year survival rate (18.5%) of HCC patients with p53 gene deletion was significantly lower than postoperative 2-year survival rate (60.0%) of those without p53 gene loss (chi(2) = 7.467, P = 0.006). Meanwhile, HER-2 oncogene amplification showed a tendency of correlation with the tumor size (chi(2) = 2.973, P = 0.085), and the postoperative 2-year survival rate (0/9) of HCC patients with HER-2 oncogene amplification was significantly lower than those (42.4%) without HER-2 oncogene amplification (chi(2) = 3.977, P = 0.046). CONCLUSION: There were a high frequency of p53 gene deletion and a low frequency of HER-2 oncogene amplification in primary HCC, which might be involved in initiation and development of a subset of primary HCC.

Adult↗

PubMed2003

Gene amplification in esophageal adenocarcinomas and Barrett's with high-grade dysplasia.

PURPOSE: The purpose of this study was to determine the frequency and overall contribution of specific gene amplification events in the formation of Barrett's adenocarcinomas. The relationship of gene amplification to clinical-pathological variables and its potential usefulness as a marker for early cancer detection were also examined. EXPERIMENTAL DESIGN: We used quantitative PCR and Southern blot analysis to screen 87 cases of Barrett's adenocarcinoma for the presence or absence of 13 distinct gene amplification events. Gene amplification was then examined for correlation with other amplification events and clinical variables (survival, stage, nodal involvement, tumor invasion, smoking history, and gender). Additionally, 22 specimens of Barrett's with high-grade dysplasia (HGD) were examined for the presence of gene amplification. RESULTS: One or more amplification events were present in 50 of 87 (57%) adenocarcinomas. The ERBB2 gene was amplified in 19 of 87 (21.8%), CCNE1 in 11 of 87 (12.6%), GATA4 in 9 of 87 (10.3%), KRAS in 9 of 87 (10.3%), EGFR in 7 of 87 (8.0%), CCND1 in 6 of 87 (6.8%), HNF3alpha in 5 of 87 (5.7%), PIK3CA in 5 of 87 (5.7%), C-MYC in 4 of 87 (4.6%), DYRK2 in 2 of 87 (2.3%), and AIB1, AKT1, and IGF1R were amplified in 0 of 87 (0%) of the tumors. CCND1 amplification was found to correlate negatively with survival (P < 0.05). In addition, the ERBB2 amplicon positively correlated (P < 0.05) with GATA4 amplification. Increased copy number of the ERBB2 (1 of 22), GATA4 (1 of 22), KRAS (2 of 22), C-MYC (1 of 22), CCNE1 (2 of 22), and CCND1 (2 of 22) genes was also observed in one or more Barrett's adenocarcinomas with HGD. CONCLUSIONS: The high frequency of gene amplification in esophageal adenocarcinomas and HGD indicates the important role of these events in esophageal adenocarcinoma development. Additionally, these results underscore the possible usefulness of early detection approaches and chemotherapeutic strategies (ErbB2 and cyclin D1) targeted against amplified gene products.

Adenocarcinoma↗

PubMed2004

Dual fluorescence in situ hybridization in detection of HER-2 oncogene amplification in primary hepatocellular carcinoma.

BACKGROUND: Molecular cytogenetics of oncogene HER-2 amplification in primary hepatocellular carcinoma (HCC) is still unknown. The aim of this study was to investigate the frequency of HER-2 oncogene amplification in primary HCC and its relations to clinicopathological parameters and prognosis. METHODS: Forty-two surgical samples from patients with primary HCC were detected for their HER-2 oncogene amplification. The number of chromosome 17 and their ratio were tested by dual fluorescence in situ hybridization (FISH) technique, and then the correlations between HER-2 amplification, clinicopathological characteristics and prognosis were analyzed statistically. RESULTS: HER-2 oncogene amplification was detected in 9 (21.4%) of the 42 primary HCCs, including 4 patients with high copy (HC) (9.5%) and 5 patients with low copy (LC) (11.9%). HER-2 amplification was associated significantly with tumor size and postoperative survival time of HCC patients (P<0.05), and the presence of HER-2 gene amplification was correlated with postoperative relapse (P=0.257), but not related to sex, age, AFP level, HBV infection, histopathological grading and clinical staging of HCC patients (P>0.05). The HER-2 oncogene copy was examined in 31 (73.8%) of the 42 primary HCCs, consisting of 9 patients with HER-2 amplification (21.4%) and 22 patients with aneuploidy (52.4%). No significant relations were observed between the HER-2 oncogene copy, patient sex, tumor size, histopathological grading, clinical staging, postoperative relapse and survival time (P>0.05); but the HER-2 oncogene copy was correlated significantly to age, AFP level and HBV infection (P<0.05). CONCLUSIONS: There are a lower frequency of HER-2 oncogene amplification and a higher frequency of chromosome 17 aneuploidy in primary HCC. HER-2 oncogene amplification may be involved in the development and progression of large HCC in some patients, and seems to be a valuably independent prognostic factor predicting the recurrence and poor survival in patients with large HCC.

Adult↗

PubMed1992

New approach for detection of amplification in cancer DNA using restriction landmark genomic scanning.

We developed a new approach for detecting the gene amplification of cancer DNAs with restriction landmark genomic scanning (RLGS). In cancer research, much effort has been made to find the amplified loci of cancer DNAs, because many lines of evidence indicate association between oncogene amplification and carcinogenesis. Conventionally, such gene amplification has been detected by using Southern hybridization with DNA probes. However, only the information of one locus can be obtained by one hybridization procedure, and analysis of many loci throughout the genome is too laborious and time consuming, even if only several candidate genes are investigated. On the other hand, the "in-gel renaturation method" was reported as another alternative for detection of amplified regions. However, even though this method is much improved, it is difficult to detect less than 7-fold amplification, which is often higher than the amplification of many cancer cases. To overcome these limitations and, in addition, to locate the amplified DNA two dimensionally, we applied RLGS for analysis of DNA amplification in cancer tissues, such as breast cancer (infiltrative tubuloadenocarcinoma), neuroblastoma, meningioma (endotheliomatous meningioma), and thyroid cancer (papillary adenocarcinoma). In some cases of breast cancer, several amplified spots located on the same amplicon were detected. In thyroid cancer, in which no amplification has yet been reported, low-grade amplification was also detected. In this report, we demonstrated that RLGS allows us to screen 2000-3000 restriction landmarks distributed on the genome simultaneously, and even low-grade amplification could be detected effectively. Thus, RLGS has proven to be a very useful method in detecting DNA amplification.

Breast Neoplasms↗

PubMed1990

myc gene amplification and expression in primary human neuroblastoma.

Although N-myc amplification in neuroblastomas correlates with poor prognosis, not all neuroblastomas which fail to respond to therapy have N-myc amplification. To determine whether other modes of myc gene activation underlie progression of some neuroblastomas, 45 were analyzed for amplification of N-myc, c-myc and L-myc and 26 were studied for transcription of these oncogenes. N-myc amplification was found in 6 of 45 tumors; no tumor had amplification of c-myc or L-myc. Transcription of both N-myc and c-myc occurred in 21 of 26 neuroblastomas. No tumor without N-myc amplification had a level of N-myc expression near that of a tumor or cell line with amplification. One tumor with N-myc amplification was the only specimen with N-myc but not c-myc expression. Five samples had c-myc but not N-myc expression; all had histological features of ganglioneuroma. DNA index did not correlate with myc gene amplification or expression. It is concluded that N-myc and c-myc are commonly expressed in primary untreated neuroblastomas, but in the absence of N-myc amplification, expression of these genes does not appear to correlate with disease progression.

Adolescent↗

PubMed1995

[Amplification of proto-oncogenes C-myc, C-N-ras, C-Ki-ras, C-erbB2 in ovarian carcinoma].

OBJECTIVE: To observe the amplification rate of C-myc, C-N-ras, C-Ki-ras, C-erbB2 in human ovarian carcinoma. METHODS: Southern blot hybridization of DNA was employed, pathological diagnosis was made from fresh tissues. The beta-globin gene was used as an internal control. RESULTS: The amplification rate of C-myc, C-N-ras, C-Ki-ras and C-erbB2 in ovarian carcinoma was 50%, 44%, 31% and 25% respectively. The amplification rates of C-myc, C-erbB2 in stage III and IV were all significantly greater than that in stage I (P < 0.01). The amplification rate of C-N-ras in stage I was also significantly greater than that in stage III (P < 0.01). The amplification rate of C-Ki-ras in stage I was significantly greater than that in stage III or IV (P < 0.01). CONCLUSIONS: The amplification of C-Ki-ras and C-N-ras took place chiefly in cases of the early stages and in cases with good differentiation. The amplification of C-N-ras was also found in cases of advanced stages. The amplifications of C-myc and C-erbB2 were chiefly found in cases above stage III and in cases with poor differentiation, 83% of the patients who died were found to have amplifications of more than 2 proto-oncogenes, with the amplification of C-erbB2 involved in all of them.

Blotting, Southern↗

PubMed1993

Chromosome 11q13, c-erbB-2, and c-myc amplification in invasive breast carcinoma: clinicopathologic correlations.

To determine the frequency and clinicopathologic correlates of chromosome 11q13 amplification in breast carcinoma, DNA from 50 invasive tumors was analyzed by Southern blot hybridization using probes for the 11q13 loci bcl-1, PRAD1, hst1, EMS1, and GST-pi, as well as oncogenes c-erbB-2 and c-myc. Sixteen tumors (32%) were amplified for one or more loci. Seven tumors (14%) showed amplification of 11q13 loci; six of these were coamplified with either c-erbB-2 (three), c-myc (two), or both (one). Nine additional tumors (18%) were amplified for c-erbB-2, and three of these were coamplified with c-myc. None showed c-myc amplification alone. Tumors with 11q13 amplification showed equivalent degrees of bcl-1, PRAD1, hst1, and EMS1 amplification, delineating an approximately 800-kb amplicon. GST-pi was inconsistently amplified, evidence that it lies outside the amplicon defined by bcl-1 and EMS1. Amplification of 11q13 was unassociated with patient age, tumor size, axillary lymph node status, hormone receptors, DNA content, histologic grade, mitotic rate, nuclear pleomorphism, or tubule formation. c-myc amplification correlated with the lack of tubule formation (p = 0.04), whereas c-erbB-2 amplification correlated with axillary lymph node positivity (p = 0.04), high histologic grade (p = 0.003), increased nuclear pleomorphism (p = 0.008), and a high mitotic rate (p = 0.02). The frequency of coamplification of 11q13 loci, c-myc, and c-erbB-2 contradicts previous proposals that amplification of these genes occurs in independent subsets of breast carcinoma. Extended clinical followup will be necessary to determine whether 11q13 amplification has prognostic utility in invasive breast cancer.

Adult↗

PubMed1996

Investigation of co-amplification of the candidate genes ornithine decarboxylase, ribonucleotide reductase, syndecan-1 and a DEAD box gene, DDX1, with N-myc in neuroblastoma. United Kingdom Children's Cancer Study Group.

Although N-myc amplification is strongly associated with a poor prognosis, not all patients with neuroblastomas having N-myc amplification fare badly. To investigate whether genes other than N-myc are responsible for contributing to the prognosis, we examined seven cell lines and 87 primary tumours for co-amplification of candidate genes known to be present near the normal N-myc locus: ornithine decarboxylase (ODC), ribonucleotide reductase (RRM2), syndecan-1 and a DEAD box protein gene, DDX1. Sequence analysis of the pG21 cDNA clone previously reported to represent an expressed gene frequently co-amplified with N-myc, showed this to be from the DDX1 gene. No co-amplification with the first three genes was found in any of the cell lines or tumour samples. DDX1, however was found to be amplified along with N-myc in 4/6 (67%) cell lines and 6/16 (38%) of the N-myc amplified tumours. Co-amplification of DDX1 and N-myc was found more frequently in stage 4 or 4S tumours than lower stage (1-3) tumours. With the exclusion of a single 4S case, there was a highly significant reduction in the mean disease-free interval from 24.4 +/- 4.7 (SE, n = 10) months for cases with co-amplification of N-myc and DDX1 compared with 9.2 +/- 1.8 (SE, n = 5) months for those cases showing amplification of N-myc alone (P = 0.0056, Welch's unpaired t-test). No amplification of DDX1, ODC, RRM2, or syndecan-1 was found in the absence of N-myc amplification. These observation indicate that the N-myc amplicon is of varied size and/or position relative to the N-myc gene, with DDX1 representing at least one other gene frequently co-amplified with N-myc. Further studies are required to confirm the biological and prognostic significance of DDX1 co-amplification and to elucidate the role that DDX1 plays in tumour genesis and progression.

Base Sequence↗

PubMed1997

Low-level c-myc amplification in human colonic carcinoma cell lines and tumors: a frequent, p53-independent mutation associated with improved outcome in a randomized multi-institutional trial.

Human colonic cancer is associated with multiple genetic deletions, mutations, and alterations in gene expression; in contrast, gene amplification has not been recognized as a prominent characteristic of human colonic tumors. Although the c-myc gene is overexpressed in approximately 70% of human colonic cancers, previous studies have not detected frequent gene amplification or rearrangement of c-myc in these tumors, although such amplification has been reported in chemically induced rodent colon cancer and quantitative analysis of gene copy number has shown the gene to be amplified at a low level in mucinous and poorly differentiated human colon carcinomas. Using rigorously controlled blot methodology, we have established that the c-myc gene, located at 8q21, exhibited amplification of 87% to 35-fold in 7 of 10 human colonic carcinoma cell lines. This was highly significant even at a low level of amplification in HT29 cells (P < 0.0001). Cytogenetic analysis by G-banding did not detect aneuploidy involving chromsome 8q, suggesting that the amplification for the c-myc gene on 8q was relatively specific, and this was consistent with a lack of amplification detected for the c-mos gene on 8q24, which was assayed similarly. The same methodology then revealed amplification of c-myc from 1.5-fold to 5-fold in 32% of tumors from 149 patients entered into a multi-institutional Phase III study of adjuvant therapy for colon cancer. c-myc status was not related to time to recurrence or death, but low levels of c-myc amplification identified a subset of patients who showed a statistically significant increase in disease-free survival, and a corresponding trend to longer overall survival, in response to adjuvant therapy with 5-fluorouracil plus levamisole. Presence of c-myc amplification was not related to incidence of p53 mutations.

Carcinoma↗

PubMed1997

Mapping of DNA amplifications at 15 chromosomal localizations in 1875 breast tumors: definition of phenotypic groups.

DNA amplification is frequent in breast cancer and has been associated with specific clinicopathological parameters and/or worsened course of the disease. In the present work, we were interested in further defining the association linking the occurrence of DNA amplification to the emergence of specific breast tumor phenotype. To this aim, we studied by Southern blotting a total of 1875 breast tumor DNAs with 26 probes mapping at 15 distinct chromosomal localizations. Of the 26 loci tested, 11 loci showed elevated levels of amplification, 9 loci showed occasional and/or low level of DNA copy number increase, and 6 loci showed very rare or no variation. This allowed us to define six amplified domains mapping at 8p12, 8q24, 11q13, 12q13, 17q12, and 20q13.2, respectively. Over 60% of the tumors analyzed presented at least one amplification at one of these localizations. Amplifications often covered large regions of DNA and bore complex patterns involving coamplification of several colocalized markers. Statistical analysis revealed correlations associating DNA amplification with breast tumor phenotype, as well as sets of preferential coamplifications. Based on these correlations, we defined three subsets of breast cancer according to their patterns of DNA amplification. The first subset (group A) was organized around the amplifications at 11q13 and/or 8p12 and was predominantly composed of estrogen receptor-positive tumors and presented a large proportion of lobular cancers. The second subset (group B) was organized around the amplifications of ERBB2 and/or MYC. These tumors were mostly estrogen receptor-negative and of the ductal invasive type. The third subset (group C) corresponded to tumors in which no amplification was detected in the present screen. Tumors in this group were largely diploid and of low histopathological grading.

Adult↗

PubMed1997

Comparative genomic hybridization analysis of breast tumors with predetermined profiles of DNA amplification.

In a separate study (F. Courjal et al., Cancer Res., 57: 4360-4367, 1997), we have analyzed by Southern blotting the relationship between DNA amplification and clinicopathological features of breast cancer. Six regions of recurrent amplifications were tested (8p12, 8q24, 11q13, 12q13, 17q12, and 20q13), and the results suggested that there was a relationship between DNA amplification profiles and breast tumor phenotype. We had delineated three subgroups of tumors showing distinct DNA amplification profiles and clinicopathological characteristics: group A, tumors showing amplification at 11q13 and/or 8p12 and/or 20q13; group B, tumors amplified at ERBB2 and/or MYC and/or MDM2/SAS; and group C, tumors with no detectable amplification. The aim of the present work was to characterize extensively the amplification profiles in the different subgroups of tumors. Sixty-one breast tumors distributed in all three subgroups were studied by comparative genomic hybridization (CGH). There was an overall good agreement between Southern blotting results and CGH data. As expected, CGH revealed gains undetected by Southern blotting. Most of these gains occurred in regions for which no adapted probes were available but also revealed nondetected amplifications at 8q24 or 20q13. Tumors showed multiple aberrations with a medium number of 5.6 copy number variations/tumor, whereas, according to Southern blotting results, 38% of the tumors analyzed were devoid of any amplification. This proportion fell to 6.5% after CGH analysis. Recurrent gains were observed in tumors from all three subgroups, albeit at varying incidences, and involved 1q, 8q, 17q23-q24, and 20q13. Gains covered large regions of DNA and could possibly include several cores of amplification. Some events, such as gains at 16p11-p12 and 14q or losses at 22q, showed more restricted distributions, suggesting the existence of additional sets of preferential coamplifications. The complexity of genetic profiles revealed by CGH indicates that breast cancer development depends on a large (yet undetermined) number of genetic events. The description of molecular phenotypes in breast cancer may therefore prove to be complex, and it should be interesting to see how many breast tumor subtypes will be defined in the end.

Adult↗

PubMedDate not supplied

Heterogeneity of MYCN amplification in a child with stroma-rich neuroblastoma (ganglioneuroblastoma).

Amplification of MYCN portends rapid tumor progression and poor prognosis in neuroblastoma. MYCN copy number has been described as homogeneous within a tumor and congruent in primary tumor and metastasis. We report a child with stage III favorable histology stroma-rich neuroblastoma (ganglioneuroblastoma) and a poor outcome with an apparent change in MYCN gene amplification by Southern blot. Initial biopsy revealed a ganglioneuroblastoma with predominance of differentiating cells designated as neuroblastoma, stroma-rich, intermixed (Shimada). Southern blot failed to demonstrate MYCN gene amplification. After front-line chemotherapy failed, a total resection was performed. In this specimen, Southern blot demonstrated MYCN amplification (15-20 copies) in the undifferentiated component and no amplification in the differentiated. Fluorescence in situ hybridization (FISH) analysis performed retrospectively on both tumor biopsies demonstrated MYCN amplification in the undifferentiated sections of both tumor specimens but not in the differentiated ones. This is the first well-documented case report of heterogeneous MYCN amplification in a child with neuroblastoma. Because key therapeutic decisions are based on the presence of MYCN amplification, physicians diagnosing and treating children with neuroblastoma need to be aware of the possibility that MYCN amplification may be heterogeneous within a tumor and may be missed using techniques based on pooled DNA such as Southern blotting. FISH may be a preferable method for determining MYCN amplification.

Blotting, Southern↗

PubMed1997

[N-myc oncogene amplification in tumors of the sympathetic nervous system: preliminary study of methods and clinico-pathologic correlations].

Tumors of the sympathetic nervous system (NT), namely neuroblastoma (NB) and ganglioneuroblastoma (GNB), have a variable clinical course. Prognostic factors include clinical stage, age of the patient, histological grade, and changes at the genomic level, of which amplification of N-myc oncogene is a well recognized phenomenon. The aim of this study was to establish the status of the N-myc oncogene in NT and to compare the results with histopathological grading, with risk groups according to criteria outlined by Joshi et al., and with the clinical stage. To detect the amplification of N-myc oncogene we used differential polymerase chain reaction (D-PCR). Amplified N-myc oncogene was found in 10 out of 31 investigated children with NB (32%). The result of D-PCR could not be interpreted reliably in two patients. Of seven children with GNB the N-myc amplification was found in one case. None of seven children with ganglioneuroma was found to have amplified N-myc oncogene. The tumors with N-myc amplification were histologically poorly differentiated of undifferentiated with a high mitotic activity-8 children were classified as a high risk category; in two we had not enough surgical material to evaluate the histological prognostic factors. The correlation of N-myc status with clinical stage revealed N-myc amplification in two of 9 children classified as clinical stage III, and in eight of 11 children classified as clinical stage IV. The N-myc amplification was not found in any child diagnosed at stage I, II and IV. Five of 10 children with NB and N-myc amplification died of the disease progression. Four children died in the group of 19 children without N-myc amplification. The median of follow-up was 18 months. The patient with GNB and N-myc amplification also died of the disease progression. Although the follow-up period of the investigated group of patients was short, the results showed that the amplification of N-myc oncogene was associated with tumors of patients diagnosed at a late clinical stage (III and IV), and with tumors whose morphology and age were assessed collectively under the term "high risk group".

Child↗

PubMed1997

The significance of HER-2/neu/c-erbB-2 gene amplification in benign and malignant breast disease.

This study was carried out to investigate the amplification of HER-2/neu oncogene in 66 patients with primary breast cancer and 90 samples from benign breast disease (BBD). The amplification of HER-2/neu oncogene in the DNA of paraffin-embedded specimens was determined by differential PCR. Nineteen out of 66 (28.8%) breast cancer patients showed amplification of the gene. No gene amplification was found in benign breast disease. There was no significant correlation of HER-2/neu amplification with, age, menopausal status, the number of positive nodes, tumor size, estrogen receptor, however, amplification of HER-2/neu gene was strongly correlated with nodal status (p = 0.0049). In node positive patients, the incidence of HER-2/neu amplification was high (43%). These findings indicate that the amplification of HER-2/neu gene may be of pathogenetic significance in breast cancer and may have a poor prognosis in node positive breast cancer patients while no gene amplification in benign breast disease suggests that HER-2/neu amplification is a late molecular alteration event in the pathogenesis of breast cancer.

Adult↗

PubMed1993

The origin of chromosome rearrangements at early stages of AMPD2 gene amplification in Chinese hamster cells.

BACKGROUND: Gene amplification and chromosomal rearrangements are frequent properties of cancer cells, provoking considerable interest in the mechanism of gene amplification and its consequences - particularly its relationship to chromosomal rearrangements. We recently studied the amplification of the gene for adenylate deaminase 2 (AMPD2) in Chinese hamster cells. Using fluorescent in situ hybridization (FISH), we found that early amplification of the AMPD2 gene is based on unequal gene segregation at mitosis, rather than local over-replication. We observed large inverted repeats of the amplified sequences, consistent with an amplification mechanism involving cycles of chromatid breakage, followed by fusion after replication and, in mitosis, the formation of bridges between the fused sister chromatids that leads to further breaks - a process we refer to as chromatid breakage-fusion-bridge (BFB) cycles. Our previous work left open the question of how this mechanism of gene amplification is related, if at all, to the chromosomal rearrangements that generate the dicentric, ring and double-minute (DM) chromosomes observed in some AMPD2-amplified metaphase cells, which are not predicted intermediates of chromatid BFB cycles, although they could be generated by related chromosome BFB cycles. RESULTS: We have addressed this question using FISH with probes for the AMPD2 gene and other markers on the same chromosome. Our results are not consistent with the chromosome BFB cycle mechanism, in which two chromatids break simultaneously and fuse to generate, after replication, a dicentric chromosome. Rather, they suggest that dicentric chromosomes are generated by secondary events that occur during chromatid BFB cycles. Our results also suggest that DM chromosomes are generated by the 'looping-out' of a chromosomal region, generating a circular DNA molecule lacking a centromere; in this case, gene amplification would result from the unequal segregation of DM chromosomes at mitosis. CONCLUSION: We conclude that, at early stages of AMPD2 gene amplification, chromatid BFB cycles are a major source of both 'intrachromosomal' gene amplification and genomic rearrangement, which are first limited to a single chromosome but which can then potentially spread to any additional chromosome. It also seems that, occasionally, a DNA sequence including the AMPD2 gene can be excised, generating a DM chromosome and thus initiating an independent process of 'extrachromosomal' amplification.

Journal Article↗

PubMed2005

[Preliminary exploration of the influence factors on amplification of single cell duplex-nested PCR].

OBJECTIVE: To explore the influence factors on amplification of single cell duplex-nested PCR. METHODS: The mutational loci region CD41-42 and IVS-II 654 of beta-globin gene were amplified by duplex-nested PCR with different combination of primers concentration, different Taq DNA polymerases, different neutralization buffers and with or without predenaturation at 98 degrees C before the PCR amplification in single lymphocyte or single blastomere, thus, to investigate the influence of these factors on the amplification efficiency of PCR. RESULTS: TaKaRa EX Taq was the most efficient Taq DNA polymerase among different Taq DNA polymerases; primer pair R1+F1 at final concentration of 0.25 micromol/L and R2+F2 at 0.3 micromol/L were the most efficient ones in amplification among different combinations of primers concentrations; the amplification efficiency in neutralization buffer-1 (200 mmol/L Tricine) was obviously higher than that of neutralization buffer-2 (900 mmol/L Tris-HCl, pH 8.3/300 mmol/L KCl/200 mmol/L HCl)(P<0.05); there were no remarkable differences of the amplification efficiency while using whether predenaturation at 98 degrees C before the single cell PCR amplification or not (P>0.05). CONCLUSION: There were remarkable differences of the amplification efficiency of single cell duplex-nested PCR while using different combination of primers concentrations, different Taq DNA polymerases, different neutralization buffers. However, predenaturation at 98 degrees C before the single cell PCR amplification could not improve the PCR amplification efficiency.

Humans↗

PubMed1993

Chromosome 11Q13 amplification in head and neck squamous cell carcinoma.

OBJECTIVE: To establish the frequency and clinicopathologic correlates of 11q13 amplification in head and neck squamous cell carcinoma. DESIGN: Retrospective clinicopathologic analysis. SETTING: University and private cancer centers. PATIENTS: Eighty-five patients with pathologically confirmed head and neck squamous cell carcinoma. MEASUREMENTS: The degree of DNA amplification in each tumor was determined using chromosome 11q13 probes for the bcl-1 major translocation cluster, PRAD1/cyclin D1 (CCND1), the fibroblast growth factor gene HST1, EMS1, and glutathione S transferase, pi-1. The presence or absence of amplification was correlated with anatomic site, tumor stage, cytologic grade, histologic pattern, and mitotic activity. RESULTS: Thirty-one patients (36%) showed a twofold to 10-fold amplification of 11q13 loci compared with the chromosome 11q23 ETS1 control probe. Twenty-nine of these encompassed bcl-1 through EMS1 loci; one sample showed only bcl-1 and PRAD1/cyclin D1 plus HST1 amplification, with another amplified at HST1 and EMS1 with minimal or no bcl-1 and PRAD1/cyclin D1 amplification. Amplification was significantly correlated with high cytologic grade, a diffusely infiltrative growth pattern, and with a hypopharyngeal primary site. CONCLUSIONS: Chromosome 11q13 amplification in head and neck squamous cell carcinoma is correlated with an aggressive histologic appearance and hypopharyngeal primary site and should be assessed in prospective clinical trials to determine its utility for treatment stratification and prognosis. Although PRAD1/cyclin D1 and EMS1 have been implicated in the pathogenesis of neoplasms with 11q13 amplification, rare cases with more limited amplicon size suggest that another relevant gene or genes may exist between these loci.

Adult↗

PubMed1997

N-myc amplification and cell proliferation rate in human neuroblastoma.

In neuroblastoma, N-myc amplification has been found to be strikingly associated with rapid tumour progression and poor prognosis. Recent studies have demonstrated that cell proliferative activity also significantly predicts the clinical outcome in patients with neuroblastoma. In order to define the correlation between N-myc amplification and cell proliferation rate, in the present investigation the two parameters were first assessed in 48 neuroblastoma tumours. N-myc amplification was evaluated in frozen specimens by Southern-blot analysis using the NB 19-21 probe and it was detected in nine patients. Cell proliferative activity was determined by measuring the AgNOR protein area in histological sections selectively stained by silver. The mean AgNOR protein area value of neuroblastomas with N-myc amplification (3.63 +/- 1.62 microns2) was not significantly different from that of neuroblastomas without N-myc amplification (2.46 +/- 1.57 microns2; P = 0.30). On the other hand, both N-myc amplification and AgNOR protein expression were found to be significantly related to the clinical outcome of the disease (P < 0.001 and P = 0.0143, respectively; median follow-up time = 47 months; range 18-106 months). In a second set of experiments, the relationship between N-myc amplification and cell proliferation rate was assessed in seven established human neuroblastoma cell lines. N-myc amplification was found to be completely independent of the population doubling time (DT), which, on the contrary, was strictly related to the quantitative expression of AgNOR protein (r = -0.947; P < 0.001). Altogether, the present results indicate that N-myc amplification and cell proliferation rate are not interrelated in neuroblastoma, each representing an independent biological parameter of cancer cells associated with the clinical behaviour of the disease.

Antigens, Nuclear↗