Study of numerical aberrations of chromosome 1 by fluorescent in situ hybridization and DNA content by densitometric analysis on (pre)-malignant cervical lesions

Summary In an attempt to determine whether the fluorescent in situ hybridization (FISH) can be used as a rapid approach for the identification of aneuploidy in premalignant cervical smears, a centromeric probe for chromosome 1 was used. The results from the FISH experiments were compared with measurements of the overall DNA content obtained by means of an image analysis system. With progression to neoplasia, a decrease of the frequency of cells with two spots was observed, due to an increasing polysomy of chromosome 1. As far as the DNA content was concerned, an increasing DNA index and 5C-exceeding ratio (fraction of cells with a DNA content higher than 5C) was observed. Classification of the FISH results by a linear discriminant analysis revealed that 67.6% of the cases were classified in agreement with the CIN classification. These data suggest that chromosome 1 may be considered as a marker chromosome for pre-malignant cervical lesions and that the DNA content measurements are complementary to the FISH results.     

The cytospectrophotometric study of the DNA content in benign and malignant processes in the breast]

Cytospectrophotometric analysis of DNA content in nuclei of the epithelial cells in fibroadenoma, fibroadenomatous and breast cancer revealed that mean value of DNA content in breast cancer is reliably higher than in benign processes in breast. Modal class in fibroadenoma and fibroadenomatous formed by di- and tetraploid cells, in breast cancer-tetraploid and higher depending on histological structure of tumor. Ratio of aneuploidy is higher in invasive ductal carcinoma and scirrhus [correction of skir] in comparison with invasive lobular carcinoma and other carcinomas, that evidences higher aggressiveness of these tumors.     

Cytofluorometric nuclear DNA content analysis of breast tissues after frozen section diagnosis

OBJECTIVE: To establish a suitable method for measurement of nuclear DNA content in breast tissues from frozen storage after frozen section diagnosis. STUDY DESIGN: For fundamental research, rat liver samples preserved in a deep freezer were used. Four protocols were used (1. fixation with 70% ethanol followed by naked nuclei preparation; 2. fixation with 10% neutral buffered formalin followed by naked nuclei preparation; 3. preparation for naked nuclei prior to fixation with 70% ethanol; and 4. preparation for naked nuclei prior to fixation with 70% neutral buffered formalin). For clinical research, 13 separate fresh frozen breast tissue samples were analyzed after frozen section diagnosis. One contained a malignant phyllodes tumor (MPT) consisting of 2 components, benign epithelial cells and malignant stromal cells; 3 were benign tumors containing fibroadenoma; and 9 cases were carcinomas, consisting of 5 scirrhous, 3 papillotubular and 1 mucinous. RESULTS: Protocols 1, 2 and 3 were not suitable methods for our purpose because remaining cytoplasm or cohesive nuclei were observed. In protocol 4 the cytoplasm was completely undetectable, and nuclei were suitably separated for nuclear DNA content measurement. Benign epithelial cell component nuclei presented a diploid pattern, and the malignant stromal cell component nuclei indicated a euploid pattern in MPT. All 3 cases of benign constituents in fibroadenoma showed a diploid pattern, as did the 3 carcinoma cases (1 mucinous, 1 scirrhous and 1 papillary). Four scirrhous and 2 papillary carcinomas showed an aneuploid pattern. CONCLUSION: Our findings show that it is possible to measure nuclear DNA content of human frozen storage tissues after frozen section diagnosis.     

The significance of DNA measurements in a histologically defined subset of infiltrating ductal carcinomas of the breast with long-term follow-up

The nuclear DNA content and other karyometric parameters were evaluated in a histologically homogeneous group of invasive ductal carcinomas of the breast from 13 patients who survived 25 years after radical mastectomy and from 13 controls matched for histologic tumor grade, lymph node status, tumor size and patient age. The nuclear DNA content and other morphometric features were evaluated by image analysis (using a modified TICAS system) on 12-microns-thick, Feulgen-stained sections. The DNA content of the tumors of both the long-term survivors and the controls varied from the diploid range to highly aneuploid (with a large proportion of the cells having a DNA content above 5N). Overall, the tumors of the controls exhibited a higher ploidy, a greater deviation from the diploid range and a greater variation of nuclear size than did the tumors of the long-term survivors. These results suggest that these measurements may be helpful in yielding prognostic information among sets of histologically identical breast tumors of similar pathologic stage.     

Centromeric repetitive DNA sequences in the genus Brassica

Representatives of two major repetitive DNA sequence families from the diploid Brassica species B. campestris and B. oleracea were isolated, sequenced and localized to chromosomes by in situ hybridization. Both sequences were located near the centromeres of many chromosome pairs in both diploid species, but major sites of the two probes were all on different chromosome pairs. Such chromosome specificity is unusual for plant paracentromeric repetitive DNA. Reduction of stringency of hybridization gave centromeric hybridization sites on more chromosomes, indicating that there are divergent sequences present on other chromosomes. In tetraploid species derived from the diploids, the number of hybridization sites was different from the sum of the diploid ancestors, and some chromosomes had both sequences, indicating relatively rapid homogenization and copy number evolution since the origin of the tetraploid species.     

Flow cytometric study of DNA distribution in cytopunctures of benign and malignant breast lesions

One hundred seventy-eight cytopunctures of mammary lesions were obtained for cytologic diagnosis and flow cytometric (FCM) analysis of the nuclear DNA content. All lesions were excised and evaluated histologically; 106 were carcinomas and 72 were benign lesions. The benign lesions showed a diploid DNA content, with one exception. Among the 106 carcinomas, 35 (33%) were diploid, 14 (13%) were tetraploid and 57 (54%) were aneuploid. For 79 carcinomas, the relationship between ploidy and (1) "T" and "N" of TNM staging, (2) the histologic grading of Scarff, Bloom and Richardson, (3) axillary nodal involvement, (4) the presence of estrogen and progesterone receptors, (5) age and (6) menopausal status was investigated. The percentage of aneuploidy was significantly higher (P less than .05) in grade III tumors as compared to grade I tumors. There was no significant relationship between aneuploidy and the other factors. However, a trend was observed between the lack of steroid receptors and a high probability of the tumor being aneuploid. FCM DNA analysis was also carried out on breast carcinomas obtained at surgery in 40 patients for whom FCM DNA analysis had previously been performed on breast cytopuncture specimens. The FCM DNA analyses were found to be best performed on the samples obtained by cytopuncture, which may increase the yield of tumor cells.     

Rapid interphase and metaphase assessment of specific chromosomal changes in neuroectodermal tumor cells by in situ hybridization with chemically modified DNA probes

Repeated DNAs from the constitutive heterochromatin of human chromosomes 1 and 18 were used as probes in nonradioactive in situ hybridization experiments to define specific numerical and structural chromosome aberrations in three human glioma cell lines and one neuroblastoma cell line. The number of spots detected in interphase nuclei of these tumor cell lines and in normal diploid nuclei correlated well with metaphase counts of chromosomes specifically labeled by in situ hybridization. Rapid and reliable assessments of aneuploid chromosome numbers in tumor lines in double hybridization experiments were achieved, and rare cells with bizarre phenotype and chromosome constitution could be evaluated in a given tumor cell population. Even with suboptimal or rare chromosome spreads specific chromosome aberrations were delineated. As more extensive probe sets become available this approach will become increasingly powerful for uncovering various genetic alterations and their progression in tumor cells.     

Relationship between cytogenetic aberrations by CGH coupled with tissue microdissection and DNA ploidy by laser scanning cytometry in head and neck squamous cell carcinoma

BACKGROUND: The relationship between DNA sequence copy number aberrations (DSCNAs) and DNA ploidy in head and neck squamous cell carcinomas (HNSCCs) is still controversial. Materials and Methods We analyzed DSCNAs by comparative genomic hybridization (CGH) combined with microdissection and DNA ploidy by laser scanning cytometry (LSC) in 18 surgically removed HNSCCs and compared the data. RESULTS: Copy number increases were most frequently observed on chromosomes 3q (16 cases), 8q (13 cases), and 12p (11 cases). Copy number decreases were observed on chromosome 3p (14 cases). LSC revealed DNA aneuploidy in 10 of the 18 cases. All DNA aneuploid tumors exhibited gain or amplification of DNA copy number at 12p11-12.1, whereas gain of DNA copy number was found in only 1 of 8 diploid tumors. DSCNAs were more frequent in DNA aneuploid tumors than in diploid tumors (P < 0.005). CONCLUSIONS: The present observations indicate a close relationship between DSCNAs and DNA ploidy in HNSCCs.     

Malignancy-associated changes in breast tissue detected by image cytometry.

In several tissues, nuclear differences have been described in normal-appearing cells from patients with invasive carcinomas compared to cases without invasive carcinoma, a phenomenon known as malignancy-associated changes (MACs). The aim of this study was to determine the presence of malignancy-associated changes in breast tissue. Image cytometry was performed on Feulgen stained tissue sections of patients with usual ductal hyperplasia with (n = 30) or without (n = 41) adjacent invasive breast carcinoma. Nuclear features of normal-appearing cells as well as of usual ductal hyperplastic cells were separately compared between the two groups. Many features of normal-appearing epithelial cells were significantly different between cases with and without invasive cancer. Significant differences were also found by measuring ductal hyperplastic nuclei instead of normal-appearing nuclei. Cases with or without cancer could be distinguished with a classification accuracy of 80% by discriminant analysis using 2 nuclear features derived from ductal hyperplastic cells. In conclusion, image cytometry on breast tissue sections shows that malignancy-associated changes can be found in normal as well as in usual ductal hyperplastic breast cells. This could be clinically relevant for the detection of occult breast cancer, for the prediction of risk in these lesions, and to monitor the effect of chemopreventive agents.     

Correlation of DNA ploidy with c-erbB-2 expression in preinvasive and invasive breast tumors.

Detection of c-erbB-2 oncogene product expression by monoclonal antibody staining (avidin-biotin technique) in formalin-fixed, paraffin-embedded atypical hyperplasias (AH, n = 20), intraductal carcinomas (IDCA, n = 27) and invasive carcinomas (INVCA, n = 48) was compared to ploidy determinations obtained by flow cytometry (INVCA) or image analysis (AH, IDCA). Cytoplasmic membrane staining was present in 11/48 (23%) INVCA and 8/27 (30%) IDCA but none of the AH. Tumors with abnormal DNA content expressed c-erbB-2 more frequently: INVCA, 2/19 (11%) diploid range versus 9/29 (31%) aneuploid; IDCA, 1/7 (14%) diploid range versus 7/20 (35%) aneuploid. Poorly differentiated (nuclear grade) IDCA or INVCA were also more frequently stained (14/35, 40%) than were well or moderately differentiated cases (5/40, 12.5%). Oncogene product expression and DNA content derangements may be related biologic parameters in breast neoplasia, and both are highly associated with cytologic nuclear abnormalities.     

Chromosome analysis in 31 cases of benign and malignant breast tumors: a study in Brazil

Cultures of 31 breast tumors, being 20 carcinomas and 11 benign lesions, were cytogenetically analysed. Clonal chromosome aberrations were detected in 16 carcinomas and in 4 benign lesions. Nine carcinomas and 2 benign lesions had multiple cytogenetically unrelated and related clones, whereas a single abnormal clone was observed in 7 carcinomas and in 2 benign lesions. Polyploid clones were found in 7 carcinomas and in 2 benign lesions. The presence of clonal chromosome aberrations and polyploid cells was not associated with the clinicopathologic parameters tested. Carcinomas had more clonal changes than benign lesions (p = 0.031), showing that cytogenetic features are of diagnostic value and that different chromosome anomalies might have different pathogenetic and prognostic significance.     

DNA nuclear content of apocrine cells in fibrocystic mastopathy and breast cancer

The DNA nuclear content of apocrine cells was studied in 5 cases of fibrocystic disease, 3 cases of invasive duct carcinoma and 3 cases of invasive lobular carcinoma. Both diploid and aneuploid values were found in each group. The possible significance of apocrine change in breast lesions is discussed.     

Identification of BCOX1, a novel gene overexpressed in breast cancer

The identification of tumor-associated antigens, which are specifically expressed in cancer tissues, is of utmost important for immunotherapy of breast cancer. We have combined in silico screening and experimental expression analysis to identify genes that are differentially expressed in breast carcinomas compared with their corresponding normal tissues. Using these approaches, we identified a novel gene, BCOX1, with overexpression in breast carcinoma. BCOX1 was highly homologous to KIAA0100, a hypothetical gene located on chromosome 17q11.2. RNA in situ hybridization shows that BCOX1 mRNA signal is mainly located in the cytoplasm of breast carcinoma epithelial cells, but not in those of normal epithelial cells, stroma cells and lymphocytes. Furthermore, mRNA expression of BCOX1 was moderately elevated in ductal in situ carcinoma (DCIS), peaked in invasive breast carcinoma (IBC) and metastatic breast carcinoma cells (MET) whereas absent in benign ductal epithelial cells. The predicted BCOX1 open reading frame of 666 bp encodes a putative protein of 222 amino acid residues with a calculated molecular weight of 2,4920 Da and a PI of 5.86. Computational analyses predict that the putative BCOX1 protein is a cytoplasmic protein. The functional relevance of this novel gene is yet to be determined. This study warrants further investigations to explore the molecular functions of BCOX1, and to determine its potential diagnostic and therapeutic applications for breast cancer.     

Epstein-Barr Virus,Human Papillomavirus and Mouse Mammary Tumour Virus as Multiple Viruses in Breast Cancer

Background

The purpose of this investigation is to determine if Epstein Barr virus (EBV), high risk human papillomavirus (HPV), and mouse mammary tumour viruses (MMTV) co-exist in some breast cancers.

Materials and Methods

All the specimens were from women residing in Australia. For investigations based on standard PCR, we used fresh frozen DNA extracts from 50 unselected invasive breast cancers. For normal breast specimens, we used DNA extracts from epithelial cells from milk donated by 40 lactating women. For investigations based on in situ PCR we used 27 unselected archival formalin fixed breast cancer specimens and 18 unselected archival formalin fixed normal breast specimens from women who had breast reduction surgery. Thirteen of these fixed breast cancer specimens were ductal carcinoma in situ (dcis) and 14 were predominantly invasive ductal carcinomas (idc).

Results

EBV sequences were identified in 68%, high risk HPV sequences in 50%, and MMTV sequences in 78% of DNA extracted from 50 invasive breast cancer specimens. These same viruses were identified in selected normal and breast cancer specimens by in situ PCR. Sequences from more than one viral type were identified in 72% of the same breast cancer specimens. Normal controls showed these viruses were also present in epithelial cells in human milk – EBV (35%), HPV, 20%) and MMTV (32%) of 40 milk samples from normal lactating women, with multiple viruses being identified in 13% of the same milk samples.

Conclusions

We conclude that (i) EBV, HPV and MMTV gene sequences are present and co-exist in many human breast cancers, (ii) the presence of these viruses in breast cancer is associated with young age of diagnosis and possibly an increased grade of breast cancer.     

Subnuclear redistribution of DNA species in confluent and growing mammalian cells

About 20 to 25 percent of the nuclear DNA from cultured cells of the African green monkey, Cercopithecus aethiops, consists of a homogeneous, highly repetitive fraction designated C. aethiops component DNA. Use of in situ hybridization techniques reveals component at the centromeres of chromosomes from both diploid and heteroploid African green monkey kidney (AGMK) tissue culture cells. — Component DNA comprises 47 percent of the nucleolar DNA in actively growing primary AGMK cells, but only 31 percent of the nucleolar DNA in confluent cells which show density-dependent growth inhibition. Further, there is a pronounced shift of both main band and component DNA from euchromatin to heterochromatin when growing cells attain confluency. Thus, the relative subnuclear distributions of component and main band DNA's are different in growing and confluent cells. — In situ hybridization techniques indicate that component sequences aggregate in clumps in nuclei of growing cells and show a diffuse distribution in nuclei of confluent cells. This suggests that centromeric regions of the various chromosomes or groups of chromosomes aggregate and disaggregate reversibly as the culture changes from density-dependent growth inhibition to active cell division. — Hypotonic citrate treatment of primary AGMK cells causes nucleoli of confluent cells to disperse: this dispersion following citrate treatment was not seen in growing AGMK cells or in confluent or growing heteroploid cells. Similarly, this nucleolar dispersion was seen in confluent diploid mouse and human cells but not in growing diploid cells or in confluent or growing heteroploid cells.     

In situ hybridization as a tool to study numerical chromosome aberrations in solid bladder tumors

Summary Methods for single- and double-target in situ hybridization (ISH) to, cells isolated from solid transitional cell carcinomas (TCC's) of the urinary bladder are described. Single cell suspensions were prepared from solid tumors of the urinary bladder by mechanical disaggregation and fixed in 70% ethanol. Using two DNA probes specific for the centromeres of chromosomes #1 and #18, ISH procedures were optimized for these samples. Human lymphocytes and cells from the T24 bladder tumor cell line were used as controls. In lymphocyte nuclei and metaphase chromosome spreads, ISH showed two major spots for each of the probes. About 80% of the nuclei from T24 cells showed three spots for both the chromosome #1 and #18 specific probes. When nuclei from TCC's were analyzed, often the number of spots for chromosome #1, and to a lesser extent for chromosome #18, differed from the number expected on basis of flow cytometric ploidy measurements. The double target-ISH method in all cases allowed the correlation of numerical aberrations for chromosomes #1 and #18 in one and the same cell. By such analyses a profound heterogeneity in chromosome number was detected in most tumors. In order to optimize the reproductbility of the method and the interpretation of the ISH-signals, criteria for their analysis have been determined. This procedure can now be applied on a routine basis to solid tumor specimens.     

Changed expression of 9-O-acetyl GD3 (CDw60) in benign and atypical proliferative lesions and carcinomas of the human breast

 Expression of gangliosides is affected in various ways by malignant cell transformation. In the present study, we investigated the expression of CDw60, a constituent of O-acetylated disialogangliosides, in benign and atypical proliferative breast diseases, and preinvasive and invasive carcinomas by immunohistochemistry and thin-layer chromatography (TLC). In normal ducts, antibodies to CDw60 (mAb M-T21) reacted to membranes of the Golgi apparatus in the juxtaluminal cell compartment. A similar polarized distribution of Golgi cisterns in epithelial cells was observed in several benign lesions, i.e., fibroadenomas, intraductal papillomas, and gynecomastia. In contrast, blunt duct adenosis and duct hyperplasia exhibited an abnormal cytosolic and cell surface staining, whereas atypical duct hyperplasia showed randomly dispersed immunoreactive Golgi cisterns, indicating loss of epithelial polarity. In mammary carcinomas and in two breast carcinoma cell lines (MCF-7 and EFM-19) the neoplastic cells contained CDw60-immunolabelled Golgi complexes, which were distributed in a disorderly fashion throughout the cytoplasm, thus reflecting a loss of epithelial polarity. Additionally, only well differentiated ductal carcinomas in situ or invasive ductal carcinomas disclosed a strong cell surface labelling, which was absent in lower differentiated carcinomas of the same types. In all carcinomas, the intensity of CDw60 immunostaining decreased with progressing loss of differentiation (grade of dedifferentiation), as demonstrated by staining intensity in paraffin sections and by evaluation of the relative amounts of extracted 9-O-acetyl GD3 by TLC. Our results indicate that abnormal CDw60 expression is already detectable in benign proliferative breast lesions with different risk rates to develop into malignant lesions. Downregulation of CDw60 expression in poorly differentiated invasive carcinomas may be the consequence of loss of cell functions usually associated with poor prognosis. Received: 19 February 1998     

DNA ploidy and vimentin expression in primary breast cancer

DNA ploidy and vimentin expression in primary breast cancer
The DNA content of 50 breast cancers of varying tumour type, grade and stage was measured using static image cytometry, and correlated with vimentin expression in the tumour cells. A tendency to increased vimentin expression and aneuploidy was observed in high grade and late stage tumours¹. A statistically significant difference was observed in DNA index and ploidy balance between grade 1 and grades 2 and 3 carcinomas ( P <0.05) and between stage I and stage II carcinomas ( P <0.05). There was a significant difference in the expression of vimentin between grades 1, 2, 3 ( P <0,001), and stages I, II and III ductal carcinomas ( P <0.05). No significant difference was observed in the proliferation index and the degree of hyperploidy ( P >0.05). Clonal heterogeneity was observed in 25% of breast carcinomas, and was associated with increased vimentin expression. These changes may be indicative of genomic alteration and tumour aggressiveness.