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The management of candidemia and disseminated candidiasis depends on rapid, unambiguous identification of Candida. Such identification is retarded by the slow growth of Candida from clinical specimens. Administration of effective but potentially toxic antifungal therapy is often withheld pending identification. To circumvent this slow growth and thus to expedite diagnosis and therapy, the polymerase chain reaction (PCR) was used to amplify a segment of fungal DNA coding for the cytochrome P450L1A1 (lanosterol-14 alpha-demethylase) in vitro. The technique provides unambiguous evidence of C. albicans in as few as 6 hours with a detection threshold of 10 organisms in a 100 mu specimen. Clinical specimens of urine (n = 4), sputum (n = 6), wound fluid (n = 1, and blood (n = 2) were collected from patients, and C. albicans was conventionally documented at these sites; in each case, PCR was confirmed. Of 17 additional specimens that were culture negative, PCR suggested the presence of yeast in two of the specimens. PCR-based detection of surgical pathogens may have broad application in rapid screening for the presence of organisms either indigenous to a particular surgical intensive care unit or peculiar to selected patient populations.
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PURPOSE: Approximately 25-30% of breast and ovarian carcinomas have amplification of the HER-2/neu oncogene. The aim of the present study was to focus on HER-2/neu gene amplification in different clinical stages of breast cancer in order to (1) determine if fluorescent in situ hybridization (FISH) can be used to detect HER-2/neu gene amplification in different clinical stages of breast cancer, (2) establish whether HER-2/neu gene amplification characterizes a subset of breast cancer in each of these stages, and (3) determine whether a trend for correlation of amplification with the clinical stage of the disease can be detected using the FISH technology. METHODS: A total of 40 specimens of formalin-fixed, paraffin-embedded breast cancer tissues were analyzed cytogenetically, in a blinded fashion, for HER-2/neu gene amplification using FISH and the Vysis LSI HER-2/neu Orange and CEP 17 Green DNA dual color probe. The criterion for "high amplification" was an amplification ratio of >4.0, that for "moderate amplification" a ratio between 2.1 and 4.0, and that for "low amplification" a ratio of 1.5-2.0. RESULTS: Using a cutoff point of > or =1.5, the overall frequency of HER-2/neu gene amplification among stage I tumors was 30% (3 out of 10). Of these, one-third (1 out of 3) showed low amplification, one-third (1 out of 3) were moderately amplified, and one-third (1 out of 3) were highly amplified. The overall frequency of HER-2/neu gene amplification among stage II tumors was 0% (0 out of 10). The overall frequency of HER-2/neu gene amplification among stage III tumors was 10% (1 out of 10). The sole tumor found positive was classified as moderately amplified by our criteria. The overall frequency of HER-2/neu gene amplification among stage IV tumors was 50% (5 out of 10). Four of the 5 tumors found positive were highly amplified. The overall frequency of gene amplification in the 40 cases studied was 22.5% (9 out of 40 tumors studied). CONCLUSION: Although a linear correlation between HER-2/neu amplification and clinical stage cannot be established at this time, it is interesting to note that when stages I and II, and when stages III and IV are combined, respectively, the latter category has a higher amplification frequency than the former. Furthermore, stage IV has the highest frequency (5 out of 10) of HER-2/neu gene amplification than all three lower stages combined (4 out of 30). This is no doubt due to the high frequency of gene amplification observed in stage IV tumors, which, interestingly, also demonstrate high level amplification of HER-2/neu gene copy numbers. Although the biologic and clinical basis for gene amplification is not clear, given the observation that the most aggressive disease stage is associated with the highest frequency of gene amplification and the most high level amplification, further exploration of HER-2/neu as a prognostic marker of poor outcome using FISH is warranted.
UNLABELLED: Deregulation of cell cycle control is a hallmark of cancer. The primary cyclins (A, B1, D1, D3 and E) are crucial for cell cycle progression. Secondary cyclins (C and H) have putative indirect effects on cell cycle propulsion and are not previously evaluated in breast cancer. We have examined protein expression and gene amplification of cyclins in breast carcinomas and correlated the findings with clinical follow-up data. We have previously demonstrated that over-expression of cyclin A is associated with poor prognosis in breast cancer patients. In this study we wanted to evaluate the mechanisms behind overexpression of cyclin A, as well as the impact of other cyclins, both at the gene level and at the protein level, on prognosis of breast cancer patients. The impact of TP53 gene mutations on gene amplification of cyclins was also evaluated. METHODS: Real-Time Quantitative PCR was used to detect gene amplification of cyclins in tumour tissue from 86 patients operated for invasive breast carcinomas, while immunohistochemistry was applied to detect protein expression of the same cyclins. RESULT: Of the 80-breast tumour samples available for cyclin A gene amplification analyses, 26.7% (23/80) was defined to have cyclin A gene amplification. 37.2% (32/79) had cyclin B1 gene amplification, 82.6% (71/82) of the samples harboured amplification of cyclin C gene, 74.4% (64/82) had cyclin D1 gene amplification, 41.9% (36/86) had cyclin D3 gene amplification, 29.1% (25/81) of the patients had cyclin E gene amplification and 9.3% (8/86) of the samples showed amplification of the cyclin H gene. When correlation between gene amplification and protein expression was evaluated, we observed a statistical significant correlation between gene amplification and protein expression of cyclin A (p=0.009) and cyclin D3 (p<0.001). However, the correlation between gene amplification and protein expression of cyclin A, as well as the prognostic value of cyclin A overexpression, was affected by gene amplification of cyclin E. Gene amplification of none of the other cyclins was associated with patient prognosis. There was a statistical significant correlation between TP53 gene mutations and gene amplification of cyclins A, D3 and B1. No correlation was observed between gene amplification of secondary cyclins (H and C) and TP53 gene mutations. CONCLUSIONS: The overexpression of cyclin A is correlated to gene amplification of both cyclin A and cyclin E. Over-expression of cyclin A is associated with poor prognosis in breast cancer patients. When analysed in a multivariate analyses model, gene amplification as well as protein expression of none of the other cyclins than cyclin A are associated with patient prognosis in breast carcinomas. TP53 gene mutation seems to correlate with gene amplification of primary, but not secondary cyclins.
Amplification of Her-2/neu in breast carcinoma is associated with poor prognosis, short disease-free interval, and short survival time in both node-negative and -positive patients. Little is known about the starting point of amplification of Her-2/neu and how it progresses from benign to malignant breast lesions. We attempted to address these questions by evaluating amplification of Her-2/neu in benign, premalignant, and malignant lesions using fluorescence in situ hybridization (FISH). Twenty-six patients with Her-2/neu-overexpressing invasive ductal carcinomas (as judged by strong immunoreactivity with Her-2/neu antibody) and coexisting lesions of ductal hyperplasia (DH), atypical ductal hyperplasia (ADH), ductal carcinoma in situ (DCIS) in the vicinity of the invasive tumor (as judged by review of the hematoxylin and eosin-stained sections), as well as metastatic carcinoma in axillary lymph nodes (mets) were selected for this study. In the primary carcinomas, a close relationship was present between overexpression as detected by immunohistochemistry (IHC) and amplification as demonstrated by FISH (85% concordance). Among these patients, amplification of Her-2/neu in ADH was demonstrated in 7 of 13 cases with ADH, and in DCIS, in 21 of 22 cases with DCIS. There was no amplification in DH or normal ductal epithelium. Significantly, in all 12 patients with synchronous positive axillary lymph nodes, there was concordant amplification of Her-2/neu in the primary and metastatic carcinoma. Amplification was consistent in multifocal metastases, despite morphological heterogeneity in some patients. Amplification ratios increased from ADH to DCIS to invasive carcinoma (P <.01, ADH versus DCIS; P <.05, DCIS versus invasive cancer), but there was no difference in amplification ratios between primary cancers and synchronous axillary metastases (P >.05). We also evaluated Her-2/neu amplification in 21 patients without Her-2/neu overexpression in their primary carcinomas (as judged by absent immunoreactivity with Her-2/neu antibody). Three showed amplification in both primary and metastatic lesions, with a low amplification ratio (approximately 2). One patient had amplification in the primary tumor but not in an axillary metastasis. Two patients exhibited slight amplification in the metastatic carcinoma (ratios 1.6 and 2), but not in their primary cancers. This FISH study indicates that amplification of Her-2/neu can emerge de novo in any stage of the disease process, from ADH to metastatic lesions, but most often appears first in ADH or DCIS. The degree of Her-2/neu amplification increases with progression to invasive carcinoma, there being no further increase in synchronous metastasis. Our data suggest that amplification of Her-2/neu appears to be mainly involved in initiation of breast oncogenesis and that its role in progression of breast cancers is uncertain.
PURPOSE: Patients with high-risk neuroblastoma who have multiple copies of MYCN fare much worse than do those without MYCN amplification; however, it has not been clarified whether intensified chemotherapy with or without blood stem cell transplantation can alter the extremely poor prognosis of patients with amplified MYCN. METHODS AND RESULTS: Between 1985 and 1999, 301 patients older than age 12 months with stage 4 neuroblastoma were treated. From January 1985 to February 1991, 80 patients with stage 4 neuroblastoma with and without MYCN amplification uniformly received induction chemotherapy with regimen A(1) (cyclophosphamide 1,200 mg/m(2) and vincristine 1.5 mg/m(2) on day 1, tetra-hydropyranyl [THP]-Adriamycin 40 mg/m(2) on day 3, and cisplatin 90 mg/m(2) on day 5). Among 22 patients with MYCN amplification, nine (40.9%) achieved a complete remission and seven (31.8%) underwent stem cell transplantation. Of 58 patients without MYCN amplification, 43 (74.1%) achieved a complete remission and 14 (24.1%) underwent stem cell transplantation. The 5-year relapse-free survival rates were 23.2% for stage 4 patients with MYCN amplification and 33.3% for those without MYCN amplification (P = 0.029); the 5-year overall survival rates were 32.8% for stage 4 patients with MYCN amplification and 42.8% for those without MYCN amplification (P > 0.05). From March 1991 to June 1998, patients with stage 4 neuroblastoma who had 10 or more copies of MYCN were treated with regimen A(3) (cyclophosphamide 1,200 mg/m(2) per day on days 1 and 2, THP-Adriamycin 40 mg/m(2) on day 3, etoposide 100 mg/m(2) per day on days 1 to 5, and cisplatin 25 mg/m(2) per day on days 1 to 5); those with fewer than 10 copies of MYCN received regimen new A (cyclophosphamide 1,200 mg/m on day 1, THP-Adriamycin 40 mg/m on day 3, etoposide 100 mg/m per day on days 1 to 5, and cisplatin 90 mg/m on day 5), which is similar in intensity to regimen A. Among 88 patients with MYCN amplification, 63 (71.6%) achieved a complete remission and 63 (71.68%) underwent stem cell transplantation. Of 133 patients without MYCN amplification, 93 (69.9%) achieved a complete remission and 71 (53.4%) underwent stem cell transplantation. The 5-year relapse-free survival rates were 36.0% for stage 4 patients with MYCN amplification and 32.2% for those without MYCN amplification (P > 0.05), the 5-year overall survival rates were 34.0% for stage 4 patients with MYCN amplification and 38.9% for those without MYCN amplification (P > 0.05). The difference in relapse-free survival rates was significantly different (P = 0.003) between patients with MYCN-amplified tumor treated before (regimen A(1)) versus after 1991 (regimen A(3)). CONCLUSIONS: With the use of the more intensive induction regimen A plus blood stem cell transplantation for MYCN-amplified patients, survival curves for those with or without MYCN amplification now appear similar. Higher doses of chemotherapy may ameliorate the effect of MYCN amplification in patients with high-risk neuroblastoma.
OBJECTIVE: To determine the clinical and prognostic significance of chromosome 11q13 amplification in squamous cell carcinoma of the head and neck. DESIGN: Retrospective clinical analysis. SETTING: University and private cancer centers. PATIENTS: Fifty-six patients with pathologically confirmed head and neck squamous cell carcinoma whose tumors had been assayed for the presence or absence of chromosome 11q13 amplification. 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 in each patient was correlated with primary site, tumor stage, nodal status, presence or absence of distant metastasis, disease recurrence, time to recurrence, clinical outcome (disease status), and overall survival. RESULTS: Amplification of chromosome 11q13 was identified in 39% (22/56) of patients. Recurrent or persistent disease was identified in 82% (18/22) of cases with amplification and 50% (14/28) of nonamplified cases (P = .04). Mean time to recurrence was shorter in cases with amplification (6.2 months) than those without amplification (10.1 months) (P = .01). Eighteen patients (82%) with amplification and 10 patients (38%) without amplification died of disease or are alive with disease (P = .001). The mean follow-up period was 15.8 months for patients with amplification and 18.6 months for patients without amplification. Overall survival was significantly diminished in patients with amplification (P = .002). Amplification was not related to nodal status, distant metastases, or initial disease stage. CONCLUSIONS: Amplification of chromosome 11q13 loci may be an important biologic marker indicating poor prognosis, independent of clinical stage in head and neck squamous cell carcinoma, and it should be assessed in prospective trials to determine its utility for stratifying treatment and determining prognosis.
The aim of this study was to assess the frequency of egfr whole gene and CA intron repeat amplification in invasive breast cancer as a mechanism for epidermal growth factor receptor (EGFR) protein overexpression. By means of tissue microarrays, protein overexpression and whole gene amplification were assessed in 222 cases of invasive breast cancer by immunohistochemistry and FISH, respectively. First intron CA repeat amplification was assessed by Taqman RT-PCR. With FISH and RT-PCR, 4.7 and 6.3% of cases showed whole gene and first intron CA repeat amplification, respectively. Amplification dosage varied between two- and four-fold in RT-PCR. By immunohistochemistry, 17.3% showed EGFR overexpression. There was a low correlation between the different methods. In all, 2.9% of cases showed both whole gene amplification and intron CA repeat amplification, and 90.3% of cases were negative for both. Nearly 20% of cases with immunohistochemical protein overexpression showed intron CA repeat amplification, and only 2.2% of cases that were negative on immunohistochemistry showed such amplification. In all, 13% of cases with protein overexpression showed amplification by FISH, and only 1.6% of cases that were negative on immunohistochemistry showed such amplification. Of the cases with EGFR overexpression, 4 (25%) showed either whole gene or intron CA repeat amplification. In conclusion, whole gene amplifications of egfr are rare in invasive breast cancer and explain protein overexpression in only about 12.5% of invasive breast cancer cases. First intron first CA repeat amplification is another important mechanism for EGFR protein overexpression, explaining protein overexpression in about 18.7% of cases. However, since about 75% of cases with EGFR protein overexpression lack either of these amplifications, other expression regulating mechanisms must be considered.
DNA amplification seems to be particularly frequent in human breast tumours and has been associated with cancer evolution and aggressiveness. Recent data indicate that new events should be added to the list, such as the amplifications at chromosome 20q13 or the MDM2 gene. The present work aimed at determining the incidence and clinicopathological signification of these amplifications in a large series of breast and ovarian tumours. We tested 1371 breast and 179 ovarian tumours by Southern blotting and observed amplification of 20q13 in 5.4% breast and 2.8% ovarian carcinomas, whereas MDM2 was found amplified in 5.3% and 3.8% of breast and ovarian tumours respectively. MDM2 RNA expression levels were analysed in a subset of 57 breast tumours and overexpression was observed in 4/57 (7%) of the tumours. Elevated expression levels coincided with amplification of the gene. In breast cancer, 20q13 and MDM2 amplifications seem to define subsets of aggressive tumours. Indeed, 20q13 was correlated to axillary nodal involvement and occurred preferentially in younger patients (< 50 years). Furthermore, 20q13 correlated, as did MDM2 amplification, to aneuploidy. In parallel, we had also tested our tumour DNAs for amplification of CCND1, ERBB-2 and MYC, which made it possible to test for correlations with 20q13 or MDM2 amplifications. Whereas 20q13 showed a very strong correlation to CCND1 amplification, that of MDM2 was prevalent in MYC-amplified tumours. Interestingly, 20q13 and MDM2 amplifications showed some degree of correlation to each other, which may possibly be owing to the fact that both events occurred preferentially in aneuploid tumours. In ovarian cancer, no statistically significant correlation was observed. However, 20q13 amplification occurred preferentially in stage 3 tumours and MDM2 was correlated to ERBB-2 amplification. This may suggest that in ovarian tumours also, 20q13 and MDM2 amplifications occur in late or aggressive cancers.
Amplification of the CDK4 gene, which encodes a key molecule in the cell cycle, has been shown in some types of human neoplasms, including bone and soft tissue tumors. It is also reported that the CDK4 gene is coamplified with other sequences in the 12q13-15 region, including the MDM2 and SAS genes. Using 146 DNA samples derived from a variety of bone and soft tissue tumors, we have studied the pattern of amplification of these three genes, CDK4, MDM2, and SAS, to investigate whether there are any tumor type specific patterns of amplification. Amplification of at least one of these three genes was found in 18 tumors, and five different patterns of amplification were observed. Amplification of all of these three genes was detected in 9 cases. Amplification of the CDK4 gene without MDM2 amplification was observed in osteosarcomas and a chondrosarcoma but not in soft tissue tumors, whereas amplification of MDM2 gene alone was observed in malignant fibrous histiocytomas (MFHs), liposarcomas, and lipomas, but not in bone tumors. These results suggested that the CDK4 region is the primary target for amplification in bone tumors, whereas the MDM2 region is in soft tissue tumors. We also investigated the relationship of CDK4 amplification with retinoblastoma (RB) gene mutations in osteosarcomas, for which we have already performed the mutation analyses in detail. Interestingly, contrary to the prevailing theory that CDK4 amplification is an alternative mechanism for RB gene mutation, we found that three of four cases with amplification of the CDK4 gene showed loss of expression of the RB protein, one of which was proved to have an gross DNA alteration in the RB locus. This redundancy of mutations may indicate that the amplification of CDK4 may have some roles other than the inactivation of the RB protein in the development of osteosarcomas.
Studies of amplification and/or overexpression of c-myc, HER-2/neu, and H-ras in breast cancer have shown that each is associated with a poor prognosis. The purpose of this study was to explore the possibility that there is a preferred sequence of amplification of these oncogenes in breast cancer. The frequencies of amplification and patterns of co-amplification of c-myc, HER-2/neu, and H-ras were studied in a group of 84 breast cancers. The data suggested a preferred sequence of amplification that consisted of c-myc amplification-HER-2/neu amplification-H-ras amplification. This model was supported by loglinear analysis. In addition, the levels of amplification of JC-A, a DNA fragment newly isolated from a patient with advanced breast cancer, were studied in 61 of these cases. The data suggested that JC-A amplification occurred early. Loglinear analysis supported a model in which JC-A amplification occurred either before or after c-myc amplification but was unrelated to Her-2/neu or ras amplification.
DNA copy number amplifications activate oncogenes and are hallmarks of nearly all advanced tumors. Amplified genes represent attractive targets for therapy, diagnostics and prognostics. To investigate DNA amplifications in different neoplasms, we performed a bibliomics survey using 838 published chromosomal comparative genomic hybridization studies and collected amplification data at chromosome band resolution from more than 4500 cases. Amplification profiles were determined for 73 distinct neoplasms. Neoplasms were clustered according to the amplification profiles, and frequently amplified chromosomal loci (amplification hot spots) were identified using computational modeling. To investigate the site specificity and mechanisms of gene amplifications, colocalization of amplification hot spots, cancer genes, fragile sites, virus integration sites and gene size cohorts were tested in a statistical framework. Amplification-based clustering demonstrated that cancers with similar etiology, cell-of-origin or topographical location have a tendency to obtain convergent amplification profiles. The identified amplification hot spots were colocalized with the known fragile sites, cancer genes and virus integration sites, but global statistical significance could not be ascertained. Large genes were significantly overrepresented on the fragile sites and the reported amplification hot spots. These findings indicate that amplifications are selected in the cancer tissue environment according to the qualitative traits and localization of cancer genes.
BACKGROUND: Global analysis of the genome, transcriptome, and proteome is facilitated by the recent development of tools for large-scale, highly parallel analysis. We describe a novel nucleic acid amplification system that generates products by several methods. 3'-Ribo-SPIA primes cDNA synthesis at the 3' polyA tail, and whole transcript (WT)-Ribo-SPIA primes cDNA synthesis across the full length of the transcripts and thus provides whole-transcriptome amplification, independent of the 3' polyA tail. METHODS: We developed isothermal linear nucleic acid amplification systems, which use a single chimeric primer, for amplification of DNA (SPIA) and RNA (Ribo-SPIA). The latter allows mRNA amplification from as little as 1 ng of total RNA. Amplification efficiency was calculated based on the delta threshold cycle between nonamplified cDNA targets and amplified cDNA. The amounts and quality of total RNA and amplification products were determined after purification of the amplification products. GeneChip array gene expression profiling and real-time PCR were used to test the accuracy and reproducibility of the method. Quantification of cDNA products (before and after amplification) at the 2 loci along the transcripts was used to assess product length (for evaluation of the 3'-initiated Ribo-SPIA) and equal representation throughout the length of the transcript (for evaluation of the whole transcript amplification system, WT-Ribo-SPIA). RESULTS: Ribo-SPIA-based global RNA amplification exhibited linearity over 6 orders of magnitude of transcript abundance and generated microgram amounts of amplified cDNA from as little as 1 ng of total RNA. CONCLUSIONS: The described methods enable comprehensive gene expression profiling and analysis from limiting biological samples. The WT-Ribo-SPIA procedure, which enables amplification of non-polyA-tailed RNA, is suitable for amplification and gene expression analysis of both eukaryotic and prokaryotic biological samples.
OBJECTIVE: To determine the degree of agreement between fluorescence in situ hybridization (FISH), Southern blot analysis and LightCycler monoplex polymerase chain reaction (PCR) analysis in the assessment of NMYC gene amplification status in neuroblastoma. STUDY DESIGN: We performed a retrospective analysis of NMYC amplification, using FISH, LightCycler monoplex PCR and Southern blot techniques to assess NMYC amplification in a series of 18 neuroblastomas and 20 histologically normal tissues (15 lymph nodes, 2 pancreas specimens, 1 section each of thyroid, prostate and uterus). RESULTS: Nine neuroblastomas were NMYC amplified, and the remaining cases were nonamplified. All cases yielded interpretable results by Southern blotting and PCR monoplexing techniques. A single case of neuroblastoma was difficult to interpret by FISH due to high background debris. A single case demonstrated low-level NMYC amplification by LightCycler PCR monoplexing but was nonamplified by the other 2 techniques. FISH analysis in 1 case showed amplification, while the other 2 techniques demonstrated nonamplified status. The case in which FISH analysis incorrectly demonstrated amplification was the same one in which there was high background debris. The Southern blot results were reported as amplified or nonamplified, while numeric amplification ratios were obtained by both FISH and PCR LightCycler monoplex analysis. Comparison of these techniques demonstrated FISH to underestimate the degree of amplification in cases in which the amplification level was high by PCR. In fact, FISH appeared to saturate at amplification ratios > 10. CONCLUSION: The study revealed a high level of concordance between the 3 techniques for assessment of NMYC amplification status. However, FISH analysis has the advantage of allowing concurrent assessment of NMYC amplification status and architecture. LightCycler PCR monoplexing appears to have the advantage of more accurately quantitating high levels of NMYC amplification, including those amplified 20-fold or higher. Both FISH and PCR LightCycler monoplexing have the advantage of being performable on formalin-fixed, paraffin-embedded tissue.