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PubMed2006

Cortactin overexpression regulates actin-related protein 2/3 complex activity, motility, and invasion in carcinomas with chromosome 11q13 amplification.

Carcinoma cell motility and invasion are prerequisites for tumor cell metastasis, which requires regulation of the actin cytoskeleton. Cortactin is an actin-related protein 2/3 (Arp2/3) complex-activating and filamentous (F)-actin-binding protein that is implicated in tumor cell motility and metastasis, partially by its ability to become tyrosine phosphorylated. Cortactin is encoded by the CTTN gene and maps to chromosome 11q13, a region amplified in many carcinomas, including head and neck squamous cell carcinoma (HNSCC). CTTN gene amplification is associated with lymph node metastasis and poor patient outcome, and cortactin overexpression enhances motility in tumor cells lacking 11q13 amplification. However, a direct link between increased motility and invasion has not been reported in tumor cells with chromosome 11q13 amplification and cortactin overexpression. In this study, we have examined the relationship between CTTN amplification and tumor cell motility in HNSCC. In 11 of 39 (28%) HNSCC cases, cortactin overexpression determined by immunohistochemistry correlates with lymph node metastasis and CTTN gene amplification. HNSCC cells containing cortactin gene amplification and protein overexpression display increased binding and activation of Arp2/3 complex, and were more motile and invasive than HNSCC cells lacking CTTN amplification. Down-regulation of cortactin expression in CTTN-amplified HNSCC cells by small interfering RNA impairs HNSCC motility and invasion. Treatment of HNSCC cells with the epidermal growth factor receptor inhibitor gefitinib inhibits HNSCC motility and down-regulates cortactin tyrosine phosphorylation. These data suggest that cortactin may be a valid prognostic and therapeutic marker for invasive and metastatic HNSCC and other carcinomas with 11q13 amplification.

Actin-Related Protein 2-3 Complex↗

PubMed2004

Reliability of t7-based mRNA linear amplification validated by gene expression analysis of human kidney cells using cDNA microarrays.

Genome wide gene expression analysis by cDNA microarrays is often limited by minute amounts of starting RNA. We therefore tested an optimized linear RNA amplification protocol using the RiboAmp amplification kit in the setting of cDNA microarrays. We isolated mRNA from a human kidney cell line (HK-2; ATCC) and from Universal Human Reference RNA (STR; Stratagene). After performing one and two rounds of linear RNA amplification, respectively, the amplified RNAs were co-hybridized to cDNA microarrays. Linearity and reproducibility of the individual experiments were then assessed by calculating the Pearson correlation. The intra-amplification consistency showed a correlation of 0.968 for the first round, 0.907 for the second round and 0.912 for two successive rounds of amplification. If the first round was compared to unamplified material, r was 0.925. The second round amplification yielded a correlation of 0.897 if compared to unamplified mRNA. Two rounds of amplification starting from 200 pg of mRNA compared to unamplified material resulted in a correlation of 0.868. These results indicate that linear amplification using RiboAmp kit yields amplified RNA with a high degree of linearity and reproducibility.

Cell Line↗

PubMed1995

Spontaneous amplification of interstitial telomeric bands in Chinese hamster ovary cells.

Chinese hamster cell lines have been widely used to explore the mechanism of gene amplification under stress conditions. Investigations of gene amplification in Chinese hamster cells under nonstress conditions, however, have been limited due to technical reasons. We initiated studies on the amplification mechanism by examining the frequency of interstitial telomeric bands (ITBs) in continuously growing cultures by fluorescence in situ hybridization (FISH), using a (TTAGGG)n probe. We found that one of the chromosomes uniquely exhibit amplification of ITBs in Chinese hamster ovary (CHO) cells but not in drug-resistant mutant CHO cells or in Chinese hamster V79 cells. Amplification of ITBs could not be induced by DNA damaging agents or DNA synthesis inhibitors. To investigate the early events that mediate ITB amplification, we developed clones and determined that the frequency of acquisition of additional ITBs on the marker chromosome was in the range of 10(-5) to 10(-3) per cell doubling. Using DNA strand-specific FISH, we found that one of the initial events of amplification is isochromosome formation. Our observations support the involvement of sister chromatid fusion in the initiation of spontaneous ITB amplification.

Animals↗

PubMed2003

Development and validation of a T7 based linear amplification for genomic DNA.

BACKGROUND: Genomic maps of transcription factor binding sites and histone modification patterns provide unique insight into the nature of gene regulatory networks and chromatin structure. These systematic studies use microarrays to analyze the composition of DNA isolated by chromatin immunoprecipitation. To obtain quantities sufficient for microarray analysis, the isolated DNA must be amplified. Current protocols use PCR-based approaches to amplify in exponential fashion. However, exponential amplification protocols are highly susceptible to bias. Linear amplification strategies minimize amplification bias and have had a profound impact on mRNA expression analysis. These protocols have yet to be applied to the analysis of genomic DNA due to the lack of a suitable tag such as the polyA tail. RESULTS: We have developed a novel linear amplification protocol for genomic DNA. Terminal transferase is used to add polyT tails to the ends of DNA fragments. Tail length uniformity is ensured by including a limiting concentration of the terminating nucleotide ddCTP. Second strand synthesis using a T7-polyA primer adapter yields double stranded templates suitable for in vitro transcription (IVT). Using this approach, we are able to amplify as little as 2.5 ng of genomic DNA, while retaining the size distribution of the starting material. In contrast, we find that PCR amplification is biased towards species of greater size. Furthermore, extensive microarray-based analyses reveal that our linear amplification protocol preserves dynamic range and species representation more effectively than a commonly used PCR-based approach. CONCLUSION: We present a T7-based linear amplification protocol for genomic DNA. Validation studies and comparisons with existing methods suggest that incorporation of this protocol will reduce amplification bias in genome mapping experiments.

Bacteriophage T7↗

PubMed2005

A robust method for the amplification of RNA in the sense orientation.

BACKGROUND: Small quantities of RNA (1-4 microg total RNA) available from biological samples frequently require a single round of amplification prior to analysis, but current amplification strategies have limitations that may restrict their usefulness in downstream genomic applications. The Eberwine amplification method has been extensively validated but is limited by its ability to produce only antisense RNA. Alternatives lack extensive validation and are often confounded by problems with bias or yield attributable to their greater biological and technical complexity. RESULTS: To overcome these limitations, we have developed a straightforward and robust protocol for amplification of RNA in the sense orientation. This protocol is based upon Eberwine's method but incorporates elements of more recent amplification techniques while avoiding their complexities. Our technique yields greater than 100-fold amplification, generates long transcript, and produces mRNA that is well suited for use with microarray applications. Microarrays performed with RNA amplified using this protocol demonstrate minimal amplification bias and high reproducibility. CONCLUSION: The protocol we describe here is readily adaptable for the production of sense or antisense, labeled or unlabeled RNA from intact or partially-degraded prokaryotic or eukaryotic total RNA. The method outperforms several commercial RNA amplification kits and can be used in conjunction with a variety of microarray platforms, such as cDNA arrays, oligonucleotide arrays, and Affymetrix GeneChip arrays.

Algorithms↗

PubMed1998

neu/erbB-2 amplification identifies a poor-prognosis group of women with node-negative breast cancer. Toronto Breast Cancer Study Group.

PURPOSE: It remains a challenge to predict which women with axillary node-negative (ANN) breast cancer at greatest risk of relapse may benefit most from adjuvant therapy. Increases in neu/erbB-2 have been implicated in breast cancer prognosis. Although overexpression has been investigated extensively, this study represents the first prospective assessment of the prognostic value of neu/erbB-2 DNA amplification in a cohort of women with newly diagnosed ANN. METHODS: A consecutive series of women was monitored for recurrence (median follow-up duration, 36 months) and tumors from 580 individuals were analyzed for amplification. The association of amplification with risk of recurrence was examined in survival analyses with traditional and histologic markers as prognostic factors. RESULTS: Neu/erbB-2 was amplified in 20% of cases. We found an increased risk of disease recurrence when neu/erbB-2 was amplified > or = twofold that persisted with adjustment for other prognostic factors (relative risk, 2.36; P = .002). We found some evidence that amplification was more important in patients who received chemotherapy compared with untreated patients. CONCLUSION: neu/erbB-2 amplification is an independent prognostic factor for risk of recurrence in ANN breast cancer. Women with tumors without neu/erbB-2 amplification have a good prognosis; aggressive therapy in this group is therefore difficult to justify. On the other hand, even with adjuvant chemotherapeutic treatment, women whose tumors exhibit neu/erbB-2 amplification have an increased risk of recurrence. We encourage a randomized trial to compare more aggressive adjuvant chemotherapy versus standard chemotherapy for ANN women whose tumors exhibit neu/erbB-2 amplification.

Axilla↗

PubMed2004

The combination of p53 mutation and neu/erbB-2 amplification is associated with poor survival in node-negative breast cancer.

PURPOSE: Increases in neu/erbB-2 have been implicated in breast cancer prognosis, but do not predict all recurrences. On the basis of evidence that p53 mutation is involved in the development of human neoplasia, we examined the prognostic value of p53 alterations in combination with neu/erbB-2 amplification. PATIENTS AND METHODS: A consecutive series of women were observed for recurrence and death (median follow-up of 85 months) and tumors from 543 individuals were analyzed for p53 mutation status and neu/erbB-2 amplification. Exons 4 through 10 of the p53 gene were analyzed by single-stranded conformational polymorphism and mutations were confirmed by DNA sequencing. The association of p53 mutation status and neu/erbB-2 amplification with risk of recurrence and death was examined in survival analyses with traditional and histologic markers as prognostic factors. RESULTS: p53 mutations occurred in 24.5% of the axillary node-negative breast carcinomas. Mutations were more frequent in carcinomas with neu/erbB-2 amplification: 38.9% compared with only 20.9% in those without neu/erbB-2 amplification. We found elevated risks of disease recurrence and overall mortality in patients with both p53 mutation and neu/erbB-2 amplification in their tumor compared with patients with neither or only one of the alterations. This increase persisted with adjustment for other prognostic factors (relative risk, 2.32; P =.002 for recurrence; relative risk, 2.22; P =.004 for death). CONCLUSION: Evaluation of tumors for p53 mutations may be beneficial to identify women at higher risk of disease recurrence and death when the tumor has neu/erbB-2 amplification, but in the absence of neu/erbB-2 amplification, the presence of p53 mutation may not provide additional independent prognostic information.

Adult↗

PubMed2002

HER2/neu amplification in breast cancer: stratification by tumor type and grade.

The presence of HER2/neu gene amplification is prognostically and therapeutically significant for patients with breast cancer. We sought to determine whether a relationship exists between HER2/neu gene amplification and the histologic type and grade of tumor. The histologic features and corresponding HER2/neu amplification results of 401 cases of invasive breast carcinoma were reviewed. Lobular carcinomas were less likely than ductal carcinomas to have HER2/neu amplification. Amplification was less frequent in Scarff-Bloom-Richardson grade I ductal carcinomas than in grades 2 and 3. Metastatic carcinomas frequently displayed HER2/neu amplification (6/20 [30%]). Our results support a correlation between HER2/neu amplification and the histologic type and grade of breast cancer. We suggest reexamination of tumors diagnosed as Scarff-Bloom-Richardson grade I invasive ductal carcinomas or lobular carcinomas if the lesion displays HER2/neu amplification to assure the exclusion of a higher grade of lesion or of missed ductal components.

Breast Neoplasms↗

PubMed2002

RNA amplification results in reproducible microarray data with slight ratio bias.

Microarray expression analysis demands large amounts of RNA that are often not available. RNA amplification techniques have been developed to overcome this prcblem, but limited data are available regarding the reproducibility and maintenance of original transcript ratios. We optimized and validated two amplification techniques: a modified in vitro transcription for the linear amplification of 3 microg total RNA and a SMART PCR-based technique for the exponential amplification of 50 ng total RNA. To determine bias between transcript ratios, we compared the expression profiles in mouse testis versus spleen between the two amplification methods and a standard labeling protocol, using microarrays containing 4596 cDNAs spotted in duplicate. With each method, replicate hybridizations were highly reproducible. However, when comparing the amplification methods to standard labeling, correlation coefficients were lower. Twelve genes that exhibited inconsistent or contradictory expression ratios among the three methods were verified by quantitative RT-PCR. The amplification methods showed slightly more discrepancies in the expression ratios when compared to quantitative RT-PCR results but were more sensitive in terms of detecting expressed genes. In conclusion, although amplification methods introduce slight changes in the transcript ratios compared to standard labeling, they are highly reproducible. For small sample size, in vitro transcription is the preferred method, but one should never combine different labeling strategies within a single study.

Blotting, Southern↗

PubMed1992

Association of epidermal growth factor receptor gene amplification with loss of chromosome 10 in human glioblastoma multiforme.

Although the loss of tumor suppressor genes and the activation of oncogenes have been established as two of the fundamental mechanisms of tumorigenesis in human cancer, little is known about the possible interactions between these two mechanisms. Loss of genetic material on chromosome 10 and amplification of the epidermal growth factor receptor (EGFR) gene are the most frequently reported genetic abnormalities in glioblastoma multiforme. In order to examine a possible correlation between these two genetic aberrations, the authors studied 106 gliomas (58 glioblastomas, 14 anaplastic astrocytomas, five astrocytomas, nine pilocytic astrocytomas, seven mixed gliomas, six oligodendrogliomas, two ependymomas, one subependymoma, one subependymal giant-cell astrocytoma, and three gangliogliomas) with Southern blot analysis for loss of heterozygosity on both arms of chromosome 10 and for amplification of the EGFR gene. Both the loss of genetic material on chromosome 10 and EGFR gene amplification were restricted to the glioblastomas. Of the 58 glioblastoma patients, 72% showed loss of chromosome 10 and 38% showed EGFR gene amplification. The remaining 28% had neither loss of chromosome 10 nor EGFR gene amplification. Without exception, the glioblastomas that exhibited EGFR gene amplification had also lost genetic material on chromosome 10 (p less than 0.001). This invariable association suggests a relationship between the two genetic events. Moreover, the presence of 15 cases of glioblastoma with loss of chromosome 10 but without EGFR gene amplification may further imply that the loss of a tumor suppressor gene (or genes) on chromosome 10 precedes EGFR gene amplification in glioblastoma tumorigenesis.

Biomarkers, Tumor↗

PubMed2003

[Detection of n-myc gene amplification in neuroblastoma using polymerase chain reaction based methods].

We have used semiquantitative and real-time quantitative PCRs to detect n-myc gene-amplification in 21 frozen neuroblastoma biopsies and IMR 32 cell line in order to predict biological behaviour of the tumors. Two primer pairs were used in the semiquantitative method to co-amplify a 520-bp fragment of the beta -globin gene -used as a single copy reference standard -and a 258-bp fragment of the n-myc gene. After 30 cycles the PCR products were electrophoresed through an agarose gel and were compared to each other with use of a gel-densitometer. Real-time quantitative analysis was performed in a LightCycler instrument. A single primer pair was used to amplify a 120-bp fragment of the n-myc oncogene and a LC640-labelled fluorescent probe pair to detect the product. Calibration curve, which was set up from a serial dilution including samples with 1, 2, 10, 13, 25-fold n-myc oncogene amplification, was employed for quantitative analysis. Semiquantitative method did not show distinct difference between tumor groups with no amplification and less than 10-fold amplification, while quantitative LightCycler analysis was able to detect even 2-fold amplification. In situ PCRs were performed in two cases of differentiated tumor samples which contained n-myc amplification. We used biotinylated ATP labelling and the same primer pair as for the LightCycler analysis.In both cases differentiated cell forms did not show n-myc gene amplification, while considerable amplification was detected in the neuroblasts.

DNA Probes↗

PubMed2000

Relating genotype and phenotype in breast cancer: an analysis of the prognostic significance of amplification at eight different genes or loci and of p53 mutations.

Breast cancer heterogeneity can be related directly to its variability at the genetic level. Thus, tumor genotyping could be a valuable approach to define breast tumor subtypes. It has been shown that it is possible to delineate subgroups of breast tumors according to specific sets of DNA amplifications. The aim of the present work was to study the prognostic significance of these DNA amplifications. We studied DNA amplification at eight genes or loci (AIB1, CCND1, EMS1, ERBB2, FGFR1, MDM2, MYC, and RMC20C001) as well as p53 mutations in a series of 640 breast cancer patients who had not received presurgical therapy and analyzed the correlations with survival DNA amplification was assessed by Southern blotting and was scored positive when exceeding three to five copies. Mutations in the p53 gene were searched by four-color fluorescent single. strand conformational polymorphism, using an automated sequencer. Of the nine genetic alterations tested, four (CCND1, EMS1, FGFR1, and p53 mutations) showed a significant association with reduced disease-free (DFS) and/or overall survival (OVS) in the unselected set of patients by univariate test. Correlations for p53 were found only when selecting mutations in exons 5 or 7. Analysis of node-negative and -positive subgroups of patients showed that MDM2 amplification and p53 mutations bore prognostic significance in node-negative patients, whereas amplification of CCND1, EMS1, and FGFR1 correlated with poor outcome in node-positive patients. Multivariate analysis on an unselected set of patients retained significance for the amplification of EMS1, FGFR1, and MDM2 with DFS, of CCND1 with OVS, and of RMC20C001 with both DFS and OVS. Interestingly, stratified analysis according to nodal status confirmed results obtained in the univariate tests: significance of MDM2 amplification and p53 mutations in node-negative and that of CCND1, EMS1, and FGFR1 in node-positive patients. We also observed an association between the number of genetic alterations observed in a tumor and poor prognosis. Patients with two or more amplified loci had a worsened outcome. Strongly correlating coamplifications such as CCND1 and FGFR1, as well as ERBB2 and MYC, were associated with a significant reduction of patient survival, thus indicating cooperative effects. Our data support the idea that genetic alterations in breast cancer are not only helpful for phenotyping purposes, but can also represent powerful prognostic indicators in the clinical practice.

Breast Neoplasms↗

PubMed2000

Gene amplifications characterize acral melanoma and permit the detection of occult tumor cells in the surrounding skin.

Acral melanoma (AM) is commonly distinguished from superficial spreading melanoma (SSM), the most common type of melanoma, by its clinical presentation as well as its ethnic distribution. However, justification for such a distinction is controversial because of histological overlap and lack of prognostic significance. We analyzed chromosomal aberrations of 15 AMs and 15 SSMs that were comparable for tumor thickness and patient age, using comparative genomic hybridization. All AMs had at least one (mean, 2.0) gene amplification, significantly more than the SSMs, in which only 2 of 15 (13%) had one amplification each (P < 0.0001). At least 15 different genomic regions were amplified in AM. These involved small portions of chromosomal arms, sometimes including known oncogenes implicated in melanoma. The most frequently amplified regions in AMs occurred at 11q13 (47%), 22q11-13 (40%), and 5p15 (20%). Comparison of the amplification levels of invasive and noninvasive portions of the tumors using fluorescence in situ hybridization suggested that amplifications occurred before the formation of the invasive portion. The finding of amplifications of 11q13 in three of five additional cases of AM in situ further supports the notion that amplifications arise early in the progression of AM. Very significantly, we found isolated melanocytes with amplifications in the epidermis up to 3 mm beyond the histologically recognizable extent of the melanomas in 5 of 15 invasive AMs. In conclusion, our data show that AM is a distinct type of melanoma characterized by focused gene amplifications occurring early in tumorigenesis, and that malignant cells are present beyond the histologically detectable boundary, thereby revealing one mechanism of local recurrence.

Chromosome Mapping↗

PubMed2000

Amplification and over-expression of the AIB1 nuclear receptor co-activator gene in primary gastric cancers.

Our analysis of chromosomal aberrations in primary gastric cancers using comparative genomic hybridization has revealed novel, high and frequent copy number increases in the long arm of chromosome 20, indicating that this region contains novel amplified genes involved in gastric cancer progression. AIB1, a member of the steroid receptor co-activator-1 family, has been cloned on 20q12 as a candidate target gene for this amplification in human breast cancers. In this study, we examined the numbers of AIB1 copies as well as their expression and relation to clinico-pathological features in 72 primary gastric cancers. AIB1 amplification was observed in 7% and over-expression in 40% of the specimens. AIB1 amplification always coincided with its over-expression, but several cases showed AIB1 over-expression without amplification, suggesting that expression of AIB1 is regulated not only by gene amplification but also by other mechanisms, such as transcriptional activation, in human gastric cancer. Gastric cancers with AIB1 amplification showed extensive lymph node metastases, liver metastases and poorer prognosis compared to those without amplification. Our results suggest that amplification and over-expression of AIB1 are likely to increase the number of malignant phenotypes of gastric cancers and that it can be expected to be useful as a marker of poor prognosis.

Adult↗

PubMed2000

Telomere sequences at the novel joints of four independent amplifications in Saccharomyces cerevisiae.

Primary gene amplification, the mutation from one copy of a gene per genome to two or more genes per genome is a major mechanism of oncogene overexpression. We previously developed a system in the yeast Saccharomyces cerevisiae to phenotypically detect primary amplifications of a reporter cassette, ADH4:CUP1. We present here the sequence analysis of novel joints from four independent, spontaneous circular amplifications identified by the ADH4:CUP1 system. All four novel joints consist of C(1-3) A telomeric repeats joined to short (14- to 16-bp) CA-rich tracts between ADH4 and the telomere of chromosome VII. In three of the four amplifications, the telomeric sequence and the CA-rich tract that are joined in the amplification are normally located in inverted orientation to each other on chromosome VII. In the fourth amplification, the CA-rich tract on chromosome VII is joined to telomere sequences from another chromosome. We suggest that formation of these amplifications was initiated by recombination between these CA-rich tracts and a telomere. The resulting dicentric chromosome could start a breakage-fusion-bridge cycle that could be resolved by the formation of a circular amplification structure.

Base Sequence↗

PubMed2001

Increased gene amplification in immortal rodent cells deficient for the DNA-dependent protein kinase catalytic subunit.

Gene amplification is one of the most frequent genome anomalies observed in tumor cells, whereas it has never been detected in cells of normal origin. A large body of evidence indicates that DNA double-strand breaks (DSBs) play a key role in initiating gene amplification. In mammals, DSBs are mainly repaired through the nonhomologous end-joining pathway (NHEJ) that requires a functional DNA-dependent protein kinase catalytic subunit (DNA-PKcs). In rodent cell lines, N-(phosphonacetyl)-L-aspartate (PALA) resistance is considered a measure of gene amplification because it is mainly attributable to amplification of the carbamyl-P-synthetase aspartate transcarbamylase dihydro-orotase (CAD) gene. In this paper we show that the radiosensitive hamster cell line V3, which is defective in DSB repair because of a mutation in the DNA-PKcs gene, displays also an increased frequency of gene amplification. In these cells, we found that the amplification of the CAD gene occurs with a frequency and a rate more than one order of magnitude higher than in control cell lines, although it relies on the same mechanisms. When the same analysis was performed in mouse embryo fibroblasts (MEFs) obtained from animals in which the DNA-PKcs gene was ablated by homologous recombination, a higher frequency of amplification compared with the controls was found only after cellular immortalization. In primary DNA-PKcs(-/-) MEFs, PALA treatment induced a block in the cell cycle, and no PALA-resistant clones were found. Our results indicate that the lack of DNA-PKcs increases the probability that gene amplification occurs in a genetic background already permissive, like that of immortalized cells, although it is not sufficient to make normal cells able to amplify.

Animals↗

PubMed2001

Amplification of HER-2/neu and topoisomerase IIalpha in primary and metastatic breast cancer.

Amplification of the HER-2/neu oncogene and amplification of the topoisomerase IIalpha gene are important determinators of the response to chemotherapy in advanced breast cancer. Assays of these genes are usually carried out using primary tumor samples, because biopsies from metastatic lesions are not usually taken. We studied the concordance of Her-2/neu and topoisomerase IIalpha amplification in primary breast tumors and their metastases by immunostaining and DNA in situ hybridization. HER-2/neu amplification, present in 28% of the primary tumors (n = 46), was always associated with amplification in its metastasis. Conversely, no metastases with HER-2/neu amplification were seen without amplification in the primary tumor. Topoisomerase IIalpha gene copy status (amplification/deletion/unaltered) remained generally unchanged in HER-2/neu-positive tumors, but in three cases, the predominant cell population in metastatic tissue was present only as a subpopulation in the primary tumor. We conclude that amplification of HER-2/neu measured in primary tumor reflects the status of metastases. Minor discrepancies between primary and metastatic tumors in topoisomerase IIalpha gene copy status may reflect evolvement of the amplicon structure in successive cell divisions.

Antigens, Neoplasm↗

PubMedDate not supplied

Expression and amplification of cyclin D1 in primary breast carcinomas: relationship with histopathological types and clinico-pathological parameters.

Overexpression and amplification of cyclin D1 were investigated by immunohistochemistry and differential polymerase chain reaction (dPCR) in 440 formalin-fixed primary breast carcinoma tissues. Overexpression of cyclin D1 was detected in 60% (263/440) and amplification of cyclin D1 was noted in 27% (119/440) of the primary breast carcinomas. Molecular analysis demonstrated that cyclin D1 was amplified in 30% (7/23) of the comedo DCIS, 22% (9/41) of the comedo DCIS and 32% (13/41) of the adjacent invasive ductal carcinomas, 30% (82/270) of the invasive ductal carcinomas, 27% (9/33) of the invasive lobular carcinomas, 19% (4/21) of the colloid carcinomas and 13% (2/15) of the medullary carcinomas. Cyclin D1 was amplified in 11% (2/19) of the invasive ductal carcinomas but not in the adjacent non-comedo DCIS lesions. Our observation showed that cyclin D1 was strongly positive in 61% (14/23) of the comedo subtype, 61% (11/18) of the non-comedo subtype, 59% (24/41) of the comedo DCIS and 63% (26/41) of the adjacent invasive ductal carcinomas, 53% (10/19) of the non-comedo DCIS and 58% (11/19) of the adjacent invasive lesions, 58% (157/270) of the invasive ductal carcinomas, 73% (24/33) of the invasive lobular carcinomas, 52% (11/21) of the colloid carcinomas and 27% (4/15) of the medullary carcinomas. A significant association was observed between in situ components and adjacent invasive lesions for cyclin D1 expression (p<0.05) and amplification (p<0.05). A significant relationship was noted between amplification of cyclin D1 and lymph node metastases (p<0.05) but not with histological grade (p>0.05), estrogen receptor status (p>0.05) and proliferation index (Ki-67 and PCNA) (p>0.05). However, overexpression of cyclin D1 was statistically associated with well differentiated tumors (p<0.05) and estrogen receptor positivity (p<0.05). No relationship was seen with nodal status (p>0.05) and proliferation index (Ki-67 and PCNA) (p>0.05). These observations suggest that tumors positive for cyclin D1 protein may have features of good prognosis but amplification of cyclin D1 gene could be an indicator of tumors with poor prognostic features. Although majority of the Malaysian patients belong to younger age group (<50 years old), amplification and expression of cyclin D1 was not statistically associated with patient age (p>0.05). These observations indicate that amplification and up-regulation of cyclin D1 may be independent of patient age. Moreover, overexpression and amplification of cyclin D1 in preinvasive, preinvasive and adjacent invasive lesions, and invasive carcinomas suggest that the gene may play an important role in early and late stages of breast carcinogenesis.

Adenocarcinoma, Mucinous↗