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PubMed2001

[Oncogene amplification and genetic heterogeneity in the metaplasia-dysplasia-adenocarcinoma sequence of Barrett esophagus].

AIMS: Information about numerical genomic alterations in the tumorigenesis of Barrett's adenocarcinoma (BCA) is still limited. In order to search for gene amplification and ploidy status, a series of locus-specific DNA probes and associated centromere probes was analysed in the metaplasia-dysplasia-adenocarcinoma-sequence. METHODS: Fluorescence in situ hybridisation (FISH) was performed on paraffin sections with locus-specific DNA probes for D7S486, c-myc, cyclin D1, Her-2/neu, 20q13.2 and associated chromosomes 7, 8, 11, 17 and 20. Corresponding areas of intestinal metaplasia (IM, n = 5), low grade dysplasia (LGD, n = 9), high grade dysplasia (HGD, n = 15) and BCA (n = 16) were analysed. RESULTS: Gene amplification of c-myc, Cyclin D1, Her-2/neu and 20q13.2 was observed in 15-35% of BCA. Coincident amplification of genes was also present. Polysomies for all investigated centromere probes were highly prevalent (up to 85%). Gene amplification was also demonstrated in HGD lesions. Polysomies were observed in HGD in high frequency (up to 80%). Extensive genetic heterogeneity was observed in both, BCA and HGD displaying different levels of amplification. None of the samples with LGD showed a locus-specific amplification, but polysomies for all investigated chromosomes were present in 18-48% of LGD. No changes were detected in BCA associated IM and squamous epithelium. CONCLUSIONS: Our data indicate that oncogene amplification of c-myc, cyclin D1, Her-2/neu, and 20q13.2 occurring in BCA and less frequently in HGD is a late event in the tumorigenesis. Polysomies of chromosomes 7, 8, 11, 17 and 20, which were highly prevalent in BCA and HGD occur already at the stage of LGD. This may be a result of an early polyploidization, preceding the later genetic events, such as gene amplification in HGD and BCA. The detection of shared numerical genomic changes and the detected extensive genetic heterogeneity in the metaplasia-dysplasia-carcinoma-sequence in Barrett's esophagus supports the hypothesis of a process of multiclonal expansion underlying this progression.

Adenocarcinoma↗

PubMed2003

Selection pressures of TP53 mutation and microenvironmental location influence epidermal growth factor receptor gene amplification in human glioblastomas.

Epidermal growth factor receptor (EGFR) gene amplification occurs in glioblastomas as so-called double minutes. Because double minutes are extrachromosomal fragments, selection pressures must operate to maintain high EGFR copy number over multiple cell divisions. In analyses of glioblastoma lysates, EGFR amplification has been observed almost exclusively in glioblastomas harboring wild-type TP53 genes, which raises the alternative hypotheses that TP53 mutation either prevents amplification or selects against maintenance of EGFR-amplified cells. To address these possibilities at the cellular level, we studied 14 glioblastomas for TP53 mutation and EGFR gene amplification status, using fluorescence in situ hybridization (FISH) for the latter. Remarkably, four of the six cases with TP53 mutation had isolated EGFR-amplified cells in different regions, demonstrating that EGFR amplification occurs frequently at the cellular level in TP53-mutant glioblastomas. Thus, TP53 mutation does not prevent EGFR amplification but does not facilitate selection of EGFR-amplified cells. Of the eight cases without TP53 mutation, five had widespread EGFR amplification. In four of these five cases, multiple regions of the tumor were available for examination; FISH demonstrated a gradation of EGFR amplification, with highly amplified cells, primarily at the invading edges rather than the relatively solid tumor centers, suggesting that EGFR overexpression, when selected for in vivo, may be related to tumor invasion.

ErbB Receptors↗

PubMed2005

Relationship between EGFR overexpression and gene amplification status in central nervous system gliomas.

OBJECTIVE: To correlate epidermal growth factor receptor (EGFR) protein overexpression, as assessed by immunohistochemistry, with EGFR gene amplification determined by fluorescence in situ hybridization in a series of gliomas. STUDY DESIGN: Forty-seven central nervous system gliomas, including 34 cases of glioblastoma multiforme, 3 oligodendrogliomas, 4 juvenile pilocytic astrocytomas and 5 low grade astrocytomas, were obtained from the files of the University of Utah Pathology Department. In each case a representative paraffin block was selected, and EGFR protein expression was quantified using immunohistochemistry. EGFR gene amplification status was determined by fluorescence in situ hybridization. RESULTS: EGFR protein overexpression was detected in 9 cases of glioblastoma multiforme. EGFR gene amplification was present in 7 of these cases. Both nonamplified glioblastomas demonstrated only 2+ overexpression of EGFR protein. None of the low grade, pilocytic or anaplastic astrocytomas demonstrated either EGFR protein overexpression or gene amplification. CONCLUSION: EGFR protein overexpression is closely associated with gene amplification. Seventy-eight percent of cases showing protein overexpression demonstrated gene amplification. All cases of central nervous system neoplasms showing protein overexpression but lacking gene amplification were associated with only 2+ protein overexpression. All central nervous system neoplasms demonstrating gene amplification and/or overexpression were high grade neoplasms.

Adult↗

PubMed1991

Variability in beta-globin and HPV DNA amplification by PCR from fixed tissues.

Paraffin-embedded tissues from a variety of sources and treated with different fixatives were tested for beta-globin and HPV amplification by polymerase chain reaction (PCR). In tests of tissues collected in the previous 2 to 3 yr, excellent rates (87% to 93%) for beta-globin amplification were obtained for specimens fixed in buffered formalin, Bouin's, and Hartmann's solutions. In contrast, the rate of beta-globin amplification was low for tissues fixed in Hollande's solution (7%) and in Hartmann's solution with eosin (33%). The results of beta-globin amplification from archival tissues stored for variable time periods showed no decrease in the amplification rate with longer periods of storage. Human papillomaviruses (HPVs) were identified in 17% of globin negative and in 43% of globin positive tissues. HPV-16 amplification was more efficient when the targeted DNA sequence was small. Variability in amplification depends not only on the type of fixative used, but also on other ill-defined factors. Therefore, conditions for optimal amplification should be determined before undertaking studies of archival material.

Blotting, Southern↗

PubMed1990

Determination of HER-2/neu amplification and expression in tumor tissue and cultured cells using a simple, phenol free method for nucleic acid isolation.

A rapid, simple and non-toxic procedure for the simultaneous isolation of DNA and RNA from tumor tissue and cells grown in vitro is described. Guanidinium isothiocyanate was used for homogenization of tumor tissue and for cell lysis. Separation of proteins, DNA and RNA was carried out by isopycnic centrifugation in cesium trifluoroacetate. DNA was further purified by salting out residual protein. Nucleic acids prepared by this method from 47 primary human carcinomas and 17 human cell lines were analysed for amplification and expression of the HER-2/neu proto-oncogene. 2- to 10-fold amplification of HER-2/neu was noted in 7/22 mammary carcinomas (32%) and in 4/14 ovarian carcinomas (28%). No amplification of the proto-oncogene was found in 4 laryngeal carcinomas, 1 pharyngeal carcinoma, 2 retrolingual carcinomas, 3 gastric carcinomas and 1 kidney carcinoma. HER-2/neu overexpression was observed in 6/22 of mammary carcinomas (27%) and 7/14 of ovarian carcinomas (50%). No overexpression was found in all other carcinomas studied. Concordance between amplification and overexpression was noted in 3 mammary and 4 ovarian carcinomas, respectively. 3 mammary and 3 ovarian carcinomas showed overexpression without amplification. 5 human mammary carcinoma cell lines showed both amplification and overexpression of HER-2/neu. In two mammary carcinoma cell lines (MDA MB-453 and ZR 75-1) overexpression was noted without amplification of the proto-oncogene. These data combine to suggest that mechanisms other than gene amplification may also lead to overexpression of the HER-2/neu protooncogene in cancer cells.

Blotting, Southern↗

PubMed1991

Oncogene amplification in breast cancer.

To refine the analysis of gene amplification in breast cancer, the authors have developed sensitive methods that can be used to screen nucleic acid prepared from a variety of sources. In their analysis, Southern hybridization and DNA dot-blot analysis were used to screen 49 breast cancer DNAs for Myc, Neu, and Int-2 gene amplification. The analysis detected minimal one extra gene copy) as well as expanded (two or more extra gene copies) gene amplifications, and in addition, distinguished between gene amplification and aneuploidy as the cause of extra gene copies. These quantitative methods were adapted to patient specimens routinely available in the anatomic pathology laboratory, including fresh tumor tissue, tumor nuclei discarded during estrogen receptor analysis, and paraffin blocks. One minimal gene amplification was found in three cases of intraductal cancer. Of 25 cases of nonmetastatic invasive cancer, 28% had at least one extra Myc gene, whereas 24% had Neu, and 21% had Int-2 gene amplification. Of 21 cases of metastatic invasive cancer, 43% had Myc, 43% had Neu, and 40% had Int-2 gene amplification. Among the nonmetastatic cancers, 47% had one, 12% had two, and 4% had three amplified genes. Within the metastatic cancers, 48% had one, 28% had two, and 5% had three amplified genes. Our data suggest relationships between tumor progression and both incidence and size of Myc, Neu, and Int-2 gene amplification.

Breast Neoplasms↗

PubMed1990

[Relationship between human papillomavirus and oncogenes (c-myc, N-myc) amplification in human cervical cancers].

The human papillomavirus detection and oncogenes amplifications were studied on DNAs from fifteen cervical cancers. We detected HPV16 and HPV18 using Southern blot hybridization and polymerase chain reaction (PCR) technique. The positive subjects of HPVs were eight cases (53%) observed by Southern blot hybridization and fourteen cases (93%) by PCR technique. The gene amplifications of oncogenes (c-myc and N-myc) were analysed by slot-blot method and were observed in c-myc but not in N-myc. The "LARGE" gene amplification (more than five fold) in c-myc was observed in one case (7%) and the "SMALL" gene amplifications (less than five fold) were observed in six cases (40%) in human cervical cancers. Although one of five cases (20%) with HPV16 was present c-myc gene amplification, all of three cases (100%) with HPV18 were found c-myc gene amplifications. In two out of three cases obtained more than three fold c-myc gene amplifications, HPV were not detectable. It is suggested that the negative correlation between gene amplification and numbers of HPV copies exist in advanced cervical cancers.

Adult↗

PubMed1989

HER-2 amplification, steroid receptors and epidermal growth factor receptor in primary breast cancer.

Amplification of HER-2 oncogene was analysed in DNAs obtained from 291 primary human mammary carcinomas. 52/291 (18%) were found to contain amplified HER-2 oncogene. Moderate amplification (2- to 5-fold) was noted in 36/291 (12%). Thirteen tumors (4.5%) had a copy number of 5 to 10. A 10- to 20-fold and greater than 20-fold amplification was observed in 2 and 1 patient, respectively. Sample sizes allowed the determination of estrogen receptor (ER) and progesterone receptor (PgR) levels in 253/291 primary breast cancers. HER-2 gene amplification was noted in 14% of ER+ patients and in 28% of ER- patients, respectively (P = 0.02). Similarly a significantly greater number of PgR- primary mammary carcinoma exhibited an amplification of the HER-2 gene compared to PgR+ cases (22% vs. 16%, P = 0.01). Although statistically not significant, tumors with HER-2 gene amplification were found to have lower levels of ER and PgR. No association of HER-2 amplification with the androgen receptor and EGF receptor was observed. Present data combine to suggest that tumor progression is more stringently controlled by the oncogene upon loss of hormone dependency. Differences found in HER-2 amplification between steroid receptor positive and negative tumors could be helpful to define a specific subset of women to whom adjuvant therapy should be directed.

Breast Neoplasms↗

PubMed1996

Expression of cyclin D1 and EMS1 in bladder tumours; relationship with chromosome 11q13 amplification.

11q13 amplifications have been found in several cancers, including bladder tumours. However, the biological significance of this genetic alteration is not yet fully understood. To get more insight into the role of 11q13 amplification in bladder tumour development, we have studied the level of amplification and expression of 4 (protoonco)genes lying within the amplicon; cyclin D1, FGF3, FGF4 and EMS1 DNA amplification was found in 5/46 tumours. There was no correlation between amplification and clinico-pathological data. No expression of FGF3 and FGF4 was detected whereas both cyclin D1 and EMS1 were expressed at higher level in tumours with amplifications. Thus cyclin D1 and EMS1, but not FGF3 and FGF4, are likely to play a pathogenic role in the 11q13 amplification in bladder cancer. However, amplification is not the unique way of activation of these genes. Indeed, in situ hybridisation and Northern blot analysis have shown that most bladder tumours have a fair to high expression of cyclin D1 and EMS1 in contrast to normal urothelium with a moderate expression. Interestingly, a trend towards higher expression occurs in superficial versus invasive tumours (8.8 +/- 2.0 versus 1.9 +/- 0.4; P approximately equal to 13% for cyclin D1 and 4.5 +/- 1.4 versus 2.0 +/- 0.4; P approximately equal to 8% for EMS1). Moreover, the 9 tumours with low expression are all highly malignant, leading to the hypothesis that the tumours developing through a cyclin D1/EMS1 independent pathway are more aggressive.

Carcinoma, Squamous Cell↗

PubMed1996

c-myc amplification in hepatocellular carcinoma predicts unfavorable prognosis.

Structural alterations and amplifications of the c-myc oncogene have been implicated in the pathogenesis and progression of several human neoplastic diseases. To study the role of c-myc amplification in human hepatocellular carcinomas (HCC), we analyzed 20 HCC using differential polymerase chain reaction (PCR). DNA used for differential PCR was extracted from formalin-fixed, paraffin-embedded tissue obtained by radiographically directed needle aspiration biopsy. Differential PCR reactions included sets of primers for c-myc and a control gene, dopamine D2 receptor (D2R), which yielded products of 150 bp and 110 bp, respectively. Evaluation of amplification was based on the relative concentration of c-myc and D2R PCR products. The c-myc amplification was detected in 10 of 20 HCC. Cases with c-myc amplification tended to have higher histologic grade and were significantly (P = 0.05) associated with worse prognosis. Amplification was present in none of two Grade 1 tumors, seven of the fourteen Grade 2 tumors, and three of four Grade 3 tumors. The mean survival times (+/- SEM) for patients with and without c-myc amplification were 5.7 (+/- 1.8) and 13.8 (+/- 2.6) months, respectively. These results indicate that c-myc amplification in HCC can be evaluated by differential PCR of needle biopsy specimens, and is an unfavorable prognostic indicator.

Adult↗

PubMedDate not supplied

Comparative amplification analysis of human glioma tissue and glioma derived fragment spheroids using reverse chromosome painting (RCP).

Tumor fragment spheroids (TFS) represent an organotypic in vitro model with preserved cytoarchitecture and matrix components of the native tumor in situ. In order to determine whether DNA amplifications within gliomas remain stable in spheroid culture, tumor fragment spheroids were established from 15 human gliomas including 14 glioblastoma and one anaplastic astrocytoma. Native tumor tissue, monolayers as well as TFS were evaluated for DNA amplification using reverse chromosome painting (RCP). A modified protocol for DNA isolation from TFS was established. Amplifications in the original tumor tissue were found on chromosomes 12q13-15, tel, 4q12-13 and 11p12-13, an amplification on 11p12-13 is reported for the first time. By RCP we could demonstrate that amplified domains on 12q13-15 and 4q12-13 in three tumors were maintained in TFS whereas the amplification on 11p12-13 could not be confirmed in TFS. In monolayer cultures, all amplifications which were detected in primary culture were lost until passage 5. The results of this first comparative study of DNA amplification in glioma by analyzing native tumor tissue and tumor fragment spheroids enables us to conclude that TFS seems to be a promising in vitro model for the study of DNA amplification under cell culture conditions.

Adult↗

PubMed1996

[Amplification of oncogenes in solid tumors in children].

BACKGROUND: The objective of the work was detection of amplification of oncogenes N-MYC, N-RAS, C-ERB A, C-ERB B and adaptor tyrosine kinase Shb in a group of 92 child age tumours in an attempt to reveal clinical and histopathological associations. METHODS AND RESULTS: Amplifications of oncogenes were detected by means of Southern's transfer, hybridization with labelled probes and densitometric evaluation. Amplification of the N-MYC oncogene in child tumours can be considered a manifestation of progression of the disease with an adverse prognosis, in particular in neuroblastomas, where it corresponds also with the adverse histological finding. In a group of sarcomas N-MYC amplification was detected in advanced clinical stages, while in malignant lymphogranulomas of the Hodgkin type it was not found. In Wilms tumour it was detected sporadically. Amplifications of oncogenes ERB A and ERB B are rare, amplifications of the oncogene RAS were not observed. Coamplifications characterized progression of the disease, in case of neuroblastoma even very short survival. In hepatic malignancies oncogene amplification was not found even in advanced stages. CONCLUSIONS: Oncogene amplification characterizes progression in a number of child tumours and its application in clinical oncology is prognostically useful.

Child↗

PubMed1998

Amplification of c-erbB-2 in gastric cancer: detection in formalin-fixed, paraffin-embedded tissue by fluorescence in situ hybridization.

A total of 396 adenocarcinomas of the stomach were examined immunohistochemically using antibodies specific for the internal domain of human c-erbB-2 protein. Forty of these (consisting of 35 well-differentiated and 5 undifferentiated types) overexpressed c-erbB-2, as evidenced by cytoplasmic membrane staining; these tumors were further examined by fluorescence in situ hybridization using a cosmid probe for 17q11.2-12 (c-erbB-2 locus) on formalin-fixed, paraffin-embedded tissues. This technique enabled us not only to detect c-erbB-2 amplification on a cell-by-cell basis, but also to compare the gene amplification with the protein expression, observe topographical distribution, and establish quantitative pictures of the amplification in vivo condition. In all 40 tumors, we observed gene amplification and found that the cancer cell with the amplification corresponded to the cells overexpressing c-erbB-2. In eight mucosal carcinomas, signals were coalesced to one or two clusters, indicating that the amplified gene resided in a homogeneous staining region (HSR). Among 32 carcinomas invading beyond muscularis mucosae, approximately half were mostly composed of cells with the amplification; the others had populations of tumor cells with and without amplification, which were sometimes in separate zones or areas and sometimes mixed together. In 22 cancers, the amplified genes were exclusively in HSR; however, cancer cells with multiple scattered signals-suggesting that the amplified genes were translocated to other chromosomes-were found exclusively in 6 tumors and concurrently with HSR in 4 tumors. These findings suggest that: (a) the amplification produces c-erbB-2 in the HSR form generally early in the carcinogenesis of gastric adenocarcinomas; and (b) in the process of cancer development, the amplified gene is lost in some cancer cells by uneven segregation of HSR in mitosis, whereas in others, it is kept in HSR or translocated to other chromosomal positions, thereby preserving overexpression of c-erbB-2 protein.

Adenocarcinoma↗

PubMed1997

Chromosome 11q13 gene amplifications in oral and oropharyngeal carcinomas: no correlation with subclinical lymph node invasion and disease recurrence.

Gene amplifications in the q13 band of chromosome 11 are among the most frequent genetic alterations in head and neck squamous cell carcinomas. Previous studies have suggested that such amplification is a marker of aggressive tumor evolution. Their potential for predicting subclinical lymph node invasion or disease recurrence was investigated in a prospective series of 50 oral and oropharyngeal carcinomas. Cell DNA content was also measured in 32 tumors of this series. Gene amplifications affecting the 11q13 band were detected in 11 of 50 (20%) patients, a relatively low frequency in comparison with data reported previously for other carcinomas of the upper aerodigestive tract, especially hypopharyngeal carcinomas. These gene amplifications were preferentially associated with aneuploidy. Cervical lymph nodes of 26 clinically N0 (Tumor-Node-Metastasis staging) patients were surgically explored. The frequency of 11q13 amplifications was very similar in the presence or in the absence of histological invasion, 3 of 15 (20%) and 2 of 11 (18%), respectively. Thus, 11q13 amplifications do not appear to be a reliable marker for prediction of subclinical lymph-node invasion in oral and oropharyngeal carcinomas. The detection of 11q13 amplifications was also not associated with a higher risk of disease recurrence. These data suggest that not only the prevalence but also the prognostic significance of 11q13 amplifications varies between tumors at different sites in the upper aerodigestive tract.

Adult↗

PubMed1995

Prognostic value of amplification of c-erb-B2 in bladder carcinoma.

Amplification of c-erb-B2 is examined in patients with transitional cell bladder carcinoma. In a pilot study we found that the amplification correlated with high tumor grade. Tumor grade is a known prognostic factor. Therefore, we next examined the prognostic value of c-erb-B2 amplification in patients with >16 years of clinical follow-up. The gene copy number was determined, using semiquantitative PCR, in archival formalin-fixed tissues. Twenty-three percent (37/163 patients) showed the amplification. The amplification correlated with grade and stage. Moreover, we found that tumor grade (P < 0.001) and c-erb-B2 amplification (P < 0.001) showed prognostic information for survival. Patients with grade 3 tumors and concomitant c-erb-B2 amplification showed the worst prognosis. Multivariate analysis indicates that grade and c-erb-B2 amplification are independent prognostic factors.

Adult↗

PubMed1995

Amplification of chromosomal region 20q13 in invasive breast cancer: prognostic implications.

Amplification of the chromosome 20q13 region was recently discovered in breast cancer by comparative genomic hybridization and subsequently further defined by fluorescence in situ hybridization with specific probes. The target gene of the amplification remains unknown. Here, fluorescence in situ hybridization with a cosmid probe for the minimal region of amplification (RMC20C001) was used to study 20q13 amplification in 132 primary breast carcinomas and 11 metastases. The size of the amplicon was studied with four flanking probes. Thirty-eight (29%) primary tumors and 3 (27%) metastases showed increased copy number of the RMC20C001 probe (>1.5-fold relative to the p-arm control). Nine (6.8%) of the primary tumors were highly (>3-fold) amplified. Although the size and location of the amplified region varied from one tumor to another, only the RMC20C001 probe was consistently amplified. 20q13 amplification was significantly associated with a high histological grade (P = 0.01), DNA aneuploidy (P = 0.01), and high S-phase fraction (P = 0.0085). High-level amplification was also associated with short disease-free survival of patients with node-negative breast cancer (P = 0.002). We conclude that high-level 20q13 amplification may be an indicator of poor clinical outcome in node-negative breast cancer and that this chromosomal region is likely to contain a gene with an important role in breast cancer progression. A large definitive study is warranted to assess the independent prognostic value of 20q13 amplification.

Adult↗

PubMed1996

Further evidence for prognostic significance of epidermal growth factor receptor gene amplification in patients with esophageal squamous cell carcinoma.

To determine the value of epidermal growth factor receptor (EGFR) gene amplification as a biological prognostic indicator in patients with esophageal squamous cell carcinoma, the EGFR gene amplification was determined by slot-blot hybridization using DNA extracted from formalin-fixed and paraffin-embedded blocks of tissues from 107 patients with esophageal squamous cell carcinoma who had undergone curative surgery. Results were analyzed by both univariate and multivariate statistical analysis. EGFR gene amplification greater than 3-fold was detected in the primary tumors of 13 (12%) of the 107 cases. The cumulative survival rate for patients with EGFR gene amplification in the primary tumors was significantly lower than that for patients without amplification (P < 0.001). A significant correlation was observed between extensive lymph node involvement at the time of surgery and EGFR gene amplification (P < 0.05). In the multivariate analysis, EGFR gene amplification (P = 0.015) and vascular invasion (P = 0.003) proved to retain independent prognostic values. These results suggest that EGFR gene amplification may be a useful biological marker for the prediction of lymph node metastasis and a poorer prognosis of esophageal squamous cell carcinoma.

Adult↗

PubMed1999

Cyclin D1 gene amplification and overexpression are present in ductal carcinoma in situ of the breast.

Cyclin D1 (CCND1) amplification is found in 10-15 per cent of invasive breast carcinomas, but it is not well established whether this gene alteration also occurs in the precursor of invasive breast carcinoma, ductal carcinoma in situ (DCIS). By Southern blot analysis, cyclin D1 gene amplification was detected in 10 per cent (3/32) of DCIS cases. In addition, 15 cases of DCIS were analysed using bright field in situ hybridization (BRISH), of which 11 had already been analysed by Southern blotting. One additional case with gene amplification was found by BRISH. The use of BRISH for the detection of gene amplification is shown to be a novel and reliable in situ method on paraffin-embedded tissue sections. By immunohistochemistry, 147 cases of DCIS were analysed for the expression of cyclin D1. Cyclin D1 overexpression was found in 9 per cent of well-differentiated, 29 per cent of intermediately differentiated, and 19 per cent of poorly differentiated DCIS. No statistically significant association was found between cyclin D1 overexpression and the differentiation grade of DCIS, although 90 per cent of the cases that show overexpression are classified as intermediately and poorly differentiated. An association was found between cyclin D1 overexpression and oestrogen receptor positivity. Cyclin D1 overexpression was found in all four cases with cyclin D1 gene amplification, but was also found in 30 per cent (8/27) of cases without detectable gene amplification. It is concluded that cyclin D1 gene amplification is an early event in the development of breast carcinoma and occurs in poorly differentiated DCIS. Cyclin D1 protein overexpression is also present in tumours without cyclin D1 gene amplification and is seen predominantly in DCIS of intermediately and poorly differentiated histological type and oestrogen receptor positivity.

Blotting, Southern↗