Genomic instability and proliferative activity as risk factors for distant metastases in breast cancer.

The role of genomic instability and proliferative activity for development of distant metastases in breast cancer was analysed, and the relative contribution of these two risk factors was quantified. A detailed quantitative comparison was performed between Ki67 and cyclin A as proliferative markers. The frequency of Ki67 and cyclin A-positive cells was scored in the same microscopic areas in 428 breast tumours. The frequency of Ki67-positive cells was found to be highly correlated with the frequency of cyclin A-positive cells, and both proliferation markers were equally good to predict risk of distant metastases. The relative contribution of degree of aneuploidy and proliferative activity as risk markers for developing distant metastases was studied independently. Although increased proliferative activity in general was associated with an increased risk of developing distant metastases, ploidy level was found to be an independent and even stronger marker when considering the group of small (T1) node negative tumours. By combining proliferative activity and ploidy level, a large group of low risk breast tumours (39%) could be identified in which only a few percentage of the tumours (5%) developed distant metastases during the 9-year follow-up time period.

Parallel cyclin E and cyclin A expression in neoplastic lesions of the uterine cervix.

Cyclin E levels are high during late G1 and early S-phase in normal cells. The cyclin E expression over the cell cycle in tumours is not fully known. The impact on patient outcome by high cyclin E levels during other parts of the cell cycle than late G1- and early S-phase is unknown. We set out to study the expression of cyclin E over the cell cycle in cervical carcinomas. Using immunofluorescence staining of cyclin A, digital microscopy, and digital image analysis, we determined which cells in a tissue section that were in S- or G2-phase. M-phase cells were detected by morphology. By simultaneously staining for cyclin E, we investigated the variation in cyclin E levels over the cell cycle in cervical carcinoma lesions. In a case-control study, in which each deceased patient was matched with a patient still alive and well after >5 years of follow-up, we found that the deceased patients had a considerably higher fraction of cyclin A-positive cells staining for cyclin E than the survivors (n = 36). We conclude that parallel cyclin E and cyclin A expression is an indicator for poor outcome in cervical carcinomas. In addition, we investigated the expression pattern of cyclin E and cyclin A in consecutive biopsy samples from cervical carcinomas at different stages, as well as in human papillomavirus positive or negative adenocarcinomas in order to further study the cyclin E and cyclin A expression pattern in neoplastic lesions of the uterine cervix.

High-resolution ROMA CGH and FISH analysis of aneuploid and diploid breast tumors.

Combining representational oligonucleotide microarray analysis (ROMA) of tumor DNA with fluorescence in situ hybridization (FISH) of individual tumor cells provides the opportunity to detect and validate a wide range of amplifications, deletions, and rearrangements directly in frozen tumor samples. We have used these combined techniques to examine 101 aneuploid and diploid breast tumors for which long-term follow-up and detailed clinical information were available. We have determined that ROMA provides accurate and sensitive detection of duplications, amplifications, and deletions and yields defined boundaries for these events with a resolution of <50 kbp in most cases. We find that diploid tumors exhibit fewer rearrangements on average than aneuploids, but rearrangements occur at the same locations in both types. Diploid tumors reflect at least three consistent patterns of rearrangement. The reproducibility and frequency of these events, especially in very early stage tumors, provide insight into the earliest chromosomal events in breast cancer. We have also identified correlations between certain sets of rearrangement events and clinically relevant parameters such as long-term survival. These correlations may enable novel prognostic indicators for breast and other cancers as more samples are analyzed.

Genetic instability promotes the acquisition of chromosomal imbalances in T1b and T1c breast adenocarcinomas.

In order to evaluate biological and genetic properties of early breast carcinomas we analyzed microdissected tissue from 33 primary breast carcinomas stage T1b and T1c with respect to the nuclear DNA content, the expression pattern of Ki-67, cyclin A, p27KIP1, p53 and p21WAF1, and chromosomal gains and losses. The results show that T1b carcinomas (6-10 mm, n=17) were frequently near-diploid (53%) with low proliferative activity and staining patterns of p53 and p21WAF1 that suggest the presence of wild type protein. The majority (12/16) of the T1c tumors (11-20 mm), however, was aneuploid, and proliferative activity and p53 expression were increased. Larger tumor size correlated with an increasing number of chromosomal copy number changes and in particular with regional amplifications. High level copy number increases (amplifications), however, were found exclusively in the aneuploid tumors. Amplification events correlated with elevated cyclin A and reduced p27 expression, respectively. Our results suggest that the sequential acquisition of genomic imbalances during tumor progression is accelerated in aneuploid tumors, and may contribute to the increased malignancy potential.

Frequent co-amplification of two different regions on 17q in aneuploid breast carcinomas.

Chromosome 17q is highly susceptible to rearrangement mutations in breast cancer. c-erbB-2 at 17q11.2 approximately q21.1 is frequently amplified, as is a region at 17q22 approximately q24. As a step in the search for the target gene(s) of the 17q22-q24 amplification we determined whether the placental lactogen (PL) genes at 17q23 were amplified in 59 breast carcinomas. These genes were selected as their upregulation could theoretically be involved in breast cancer tumorigenesis. Amplification of the PL genes, and also of c-erbB-2, was detected using semi-quantitative PCR. The reliability of this method was confirmed since c-erbB-2 results obtained using PCR, Southern blotting and immunohistochemistry were in good agreement. The PL genes were amplified in 13 (22%) of the tumors. Furthermore, the PL and c-erbB-2 genes were frequently co-amplified although there is a non-amplified region between them. Expression of PL was investigated in 26 tumors and was detected in 16 of these cases including all 10 tumors with amplification of the PL genes. The tumors with PL gene amplification were all aneuploid. A trend was seen towards an increased incidence of lymph node involvement for tumors with amplification of the PL genes and for tumors with co-amplification of PL and c-erbB-2, which suggests a possible association with high malignancy.

Accumulation of cyclin E is not a prerequisite for passage through the restriction point.

The restriction point (R) is defined as the point in G(1) after which cells can complete a division cycle without growth factors and divides G(1) into two physiologically different intervals in cycling cells, G(1)-pm (a postmitotic interval with a constant length of 3 to 4 h) and G(1)-ps (a pre-DNA-synthetic interval with a variable length of 1 to 10 h). Cyclin E is a G(1) regulatory protein whose accumulation has been suggested to be critical for passage through R. We have studied cyclin E protein levels in individual cells of asynchronously growing cell populations, with respect to both passage through R and entry into S phase. We found that the postmitotic G(1) cells that had not yet reached R were negative for cyclin E accumulation. On the other hand, cells that had passed R were found to accumulate cyclin E at variable times (1 to 8 h) after passage through R and 2 to 5 h before entry into S. These kinetic data rule out the hypothesis that passage through R is dependent on the accumulation of cyclin E but suggest, instead, the converse, that passage through R is a prerequisite for cyclin E accumulation. Furthermore, we found that most of the cyclin E protein is downregulated within 1 to 2 h after entry into S.

Frequent amplification of the telomerase reverse transcriptase gene in human tumors.

Activation of telomerase is a crucial step during cellular immortalization and malignant transformation of human cells and requires the induction of the catalytic component, human telomerase reverse transcriptase (hTERT), encoded by the hTERT gene. It is poorly understood how the hTERT gene is activated in human cancer cells. In the present study, we examined the hTERT gene copy number in human cancer cell lines and in primary tumor tissues. Amplification of the hTERT gene was observed in 8 of 26 (31%) tumor cell lines and 17 of 58 (30%) primary tumors examined (8 of 21 lung tumors, 3 of 10 cervical tumors, 5 of 19 breast carcinomas, and 1 of 8 neuroblastomas). In addition, 13 of 26 (50%) cell lines and 13 of 58 (22%) primary tumors displayed gain of hTERT gene copies with 3-4 copies/cell. The present findings imply that the hTERT locus may be a frequent target for amplification during tumorigenesis and that this genetic event probably contributes to the dysregulation of telomerase activity occurring in human tumors.

Overexpression of MYC causes p53-dependent G2 arrest of normal fibroblasts.

Overexpression of the proto-oncogene MYC has been implicated in the genesis of diverse human cancers. One explanation for the role of MYC in tumorigenesis has been that this gene might drive cells inappropriately through the division cycle, leading to the relentless proliferation characteristic of the neoplastic phenotype. Herein, we report that the overexpression of MYC alone cannot sustain the division cycle of normal cells but instead leads to their arrest in G(2). We used an inducible form of the MYC protein to stimulate normal human and rodent fibroblasts. The stimulated cells passed through G(1) and S but arrested in G(2) and frequently became aneuploid, presumably as a result of inappropriate reinitiation of DNA synthesis. Absence of the tumor suppressor gene p53 or its downstream effector p21 reduced the frequency of both G(2) arrest and aneuploidy, apparently by compromising the G(2) checkpoint control. Thus, relaxation of the G(2) checkpoint may be an essential early event in tumorigenesis by MYC. The loss of p53 function seems to be one mechanism by which this relaxation commonly occurs. These findings dramatize how multiple genetic events can collaborate to produce neoplastic cells.

A detailed analysis of cyclin A accumulation at the G(1)/S border in normal and transformed cells.

The temporal relationship between cyclin A accumulation and the onset of DNA replication was analyzed in detail. Five untransformed and nine transformed asynchronously growing cell cultures were investigated using a triple immunofluorescence staining protocol combined with computerized evaluation of staining intensities in individual cells. The simultaneous staining of BrdU, cyclin A, and cyclin E made it possible to determine the cell cycle position of each cell investigated. Cells at the G(1)/S border were identified on the basis of cyclin E content and were further analyzed with respect to cyclin A and BrdU content. A method was developed to calculate objective thresholds defining the highest staining intensity found in the negative cells in the population. Using the thresholds we could distinguish cells with minute amounts of cyclin A and BrdU from truly negative cells. We show that the onset of cyclin A accumulation and the start of DNA replication occurs at the same time, or deviating by a few minutes at the most. We also show that cyclin A accumulates continuously during S. This study clearly demonstrates that nuclear cyclin A can be used as a reliable marker for the S and G(2) phases in both normal and transformed interphase cells.

Reliability and significance of DNA measurements in interphase nuclei and division figures in histological sections.

DNA contents from single cells at interphase and division were analysed in histological sections and in imprints from 73 breast cancer specimens. Fetal livers from 18 terminations of normal pregnancies provided the standard for truly mitotic prophases, metaphases and telophases. The reliability of DNA quantities from image microphotometry was improved using paraffin-embedded tissue samples from which 4, 8 and 15 microns slices were Feulgen stained. Imprinted replicas from the mirror surface of each freshly cut specimen provided matching domains and represent the crucial approach in this project. A close positive relationship was observed between interphase nuclei in 8 microns sections and their imprinted counterparts (r = 0.992; n = 73). Interphase nuclei in 4 microns sections yielded insufficient DNA contents when compared with the imprints (r = 0.815; n = 21) and with endogenous lymphocyte nuclei. This 2 cDNA standard also calibrated 232 mitotic figures to 3.91 +/- 0.01 c in 15 microns sections from fetal liver. Prophases, metaphases and telophases were slightly scattered (coefficient of variation = 0.04 each). The 0.09 c deficiency to plain 4.0 c was read as an artifact from sectioning. However, the methodical bias did not challenge the most irregular DNA distribution profiles recorded from chromosome division figures (CDFs) in 15 microns sections of breast cancers. Poorly differentiated and aggressive breast cancer (Auer type IV, Zetterberg type A) exhibited a 4.5 c exceeding rate of 82.24% from a total of 752 CDFs in 10 randomly selected cases. Well differentiated, slowly growing cancer with diploid interphase nuclei (Auer I, Zetterberg D) surprisingly showed a 4.5 c exceeding rate of 29.26% from a total of 173 mitoses and CDFs in 10 randomly selected cases. The bulk of data beyond the mitotic 4.0 c level discriminates biological bias from methodical impairment. We concluded that 8 microns sections are sufficient for human interphase nuclei, whereas a depth of 15 microns preserves intact mitoses and CDFs.

Analysis of mechanisms and frequency of CDKN2A/B gene loss during progression of RAS-transformed rat embryo fibroblast clones.

Rat embryo fibroblasts (REFs) are inefficiently transformed by RAS-oncogenes. Induction of p16INK4A expression by RAS has been suggested to contribute to this resistance. Glucocorticoid hormones, (DEX), enhance REF transformation by RAS and facilitates the isolation of transformed and immortal cell lines. We show that DEX induced cell proliferation is paralleled by a decrease in Cdkn2a gene transcripts, suggesting a mechanism for hormone promotion. The mechanisms of progression into hormone independent cell lines were examined. Twenty-two of 30 clones which reached a population size of approximately 10(6) cells could be established as cell lines. All lines studied showed homozygous deletions of the Cdkn2 loci (Cdkn2a and Cdkn2b) on RNO5. LOH was found for all RNO5 genetic markers examined in 7 of 19 cell lines, suggesting non-disjunction events. In the remaining 12 cell lines, both copies of Cdkn2 appeared to be lost by deletions/rearrangements, some of which could by demonstrated by karyotype analysis. We conclude that (i) clonal expansion of RAS-transfected REF by DEX is paralleled by down-regulation of Cdkn2a expression; (ii) homozygous deletion of Cdkn2 were estimated to occur at a frequency of 2 x 10(-8)/cell/generation or higher, and (iii) deletion/rearrangements and nondisjunction appear to be the main mechanisms leading to deletion of Cdkn2.

Near tetraploid prostate carcinoma. Methodologic and prognostic aspects.

BACKGROUND: The clinical value of DNA ploidy analysis in prostate carcinoma has been an issue for investigation for more than 2 decades. In general, diploid or pseudodiploid tumors are associated with a favorable prognosis and aneuploid tumors with an unfavorable prognosis, irrespective of type of treatment. Tumors with DNA values in the tetraploid region (around 4c) present a diagnostic problem. Such DNA distributions may clearly represent aneuploid tumors with an unfavorable prognosis. However, a 4c distribution may conversely represent a tetraploid tumor (possibly a polyploid variant of the diploid tumor) with a favorable prognosis. Previous data from our laboratory indicate the existence of such a tetraploid subgroup. The goal of the current study was to investigate the diagnostic problem of 4c tumors in greater detail. METHODS: Ploidy classification of cytologic smears by image cytometry was performed in a retrospective study of 334 patients with hormonally treated prostate carcinoma. Follow-up time was 30 years or until death. RESULTS: Three ploidy types were defined: near-diploid (D type), near-tetraploid (T type), and highly aneuploid (A type). Tumors with a modal value within the tetraploid region were found in 27% (92 cases) of the total material. Of these, 9% were defined as T type and 18% as A type. Overall, 37% of the tumors were classified as D type, 9% as T type, and 54% as A type. Of the A type tumors, one-third had modal DNA values in the tetraploid (4c) region. Multivariate analysis showed a statistically significant difference between A type tumors and D and T type, but not between D type and T type. Both D and T type tumors progressed slowly and killed the patients 5 to 30 years after diagnosis, whereas A type tumors progressed rapidly and killed the patients within 6 years of diagnosis. CONCLUSIONS: By image cytometry, prostate carcinoma can be divided into three ploidy types: D, T, and A type. Biologically, however, the tumors fall into only two groups: low grade malignant, pseudodiploid tumors of D or T type, and high grade malignant, highly aneuploid tumors of A type.

Molecular characterization of a tissue-polypeptide-specific-antigen epitope and its relationship to human cytokeratin 18.

Tissue-polypeptide-specific antigen (TPS) from the human colon adenocarcinoma cell line WiDr was purified using the monoclonal antibody M3 as a probe. Upon SDS/polyacrylamide gradient gel electrophoresis, several TPS-positive bands were detected (corresponding to 13 kDa, 22 kDa and a doublet at 42 kDa). The 13-kDa moiety was purified about 30,000-fold by a 5-step protocol. The electro-phoretically homogeneous component was obtained in a 7% yield of the total TPS activity of the crude extract. N-terminal sequence analysis showed the presence of an N-terminally truncated molecule and identified the 13-kDa TPS component as a fragment of human cytokeratin 18, with a major from starting at position 284 of the parent molecule. Laser-desorption mass spectrometry showed the presence of one major component with a molecular mass corresponding to a C-terminal end close to position 396 (which gives 12776 Da for the form with non-truncated N-terminus). The M3 antibody was also used to screen a human prostate cDNA lambda gt11 library. Four identical phage clones were detected, each producing a fusion protein with beta-galactosidase and the M3-positive component. PCR amplification showed the presence of an approximately 1200-bp insert, and sequence analysis revealed it to contain a 996-nucleotide fragment corresponding to residues 103-429 of human cytokeratin 18 (plus a non-coding human desmin artifact fragment). Smaller fragments, engineered by PCR and expressed as fusion proteins using the pET3xc vector in Escherichia coli, showed that the M3 epitope is localized to cytokeratin 18, residues 322-340. Two other TPS-active monoclonal antibodies were localized to cytokeratin 18 with similar techniques, ascribing an epitope (to M21) to residues 414-429 and another (to M24) to residues 139-297. Combined, the results demonstrate that TPS reactivity is derived from specific epitopes of human cytokeratin 18.

Chromosomal localization and 5' sequence of the human protein serine/threonine phosphatase 5' gene.

Protein phosphorylation plays a crucial role in the regulation of a wide array of proteins involved in many cellular processes. Protein phosphatase 5 (PP5) is a novel member of the protein serine/threonine phosphatase family. The majority of the cDNA sequence of PP5 has been reported recently. In our study, a sequence encoding the whole open reading frame of PP5 was cloned from a human fetal brain cDNA library. The protein phosphatase cDNA sequence of our clone is longer at the 5' end than the recently published sequence. It's likely that the extended sequence contains the start codon ATG, since a translation stop codon TAG is present upstream of the ATG codon in the same open reading frame. The mRNA of the PP5 gene was detected in all the human tissues examined. The PP5 gene was localized to human chromosomal region 19q13.3.

Relationship between oncogene amplification, aneuploidy and altered expression of p53 in breast cancer.

Many studies have noted an association between amplification of single oncogenes and poor prognosis in breast cancer. We are investigating whether measurement of amplification in a larger number of proto-oncogenes increases the reliability of the prognostic information provided. As the first stage of this investigation, amplification (of c-erbB-2, cycD1, int-2, c-myc and MDM2), aneuploidy and altered expression of p53, which all indicate genetic instability, were studied in 117 primary breast adenocarcinomas. Amplification was correlated with aneuploidy (p=0.002) but not with altered expression of p53 even though the tumours with p53 overexpression were all aneuploid. Our results suggest that measurement of amplification is a potentially valuable prognostic factor.

What is the restriction point?

The restriction point (R) separates two functionally different parts of G1 in continuously cycling cells. G1-pm represents the postmitotic interval of G1 that lasts from mitosis to R. G1-ps represents the pre S phase interval of G1 that lasts from R to S. G1-pm is remarkably constant in length (its duration is about three hours) in the different cell types studied so far. G1-ps, however, varies considerably, indicating that entry into S is not directly followed after passage through R. Progression through G1-pm requires continuous stimulation by mitogenic signals (e.g. growth factors) and a high rate of protein synthesis. Interruption of the mitogenic signals or moderate inhibition of protein synthesis leads to a rapid exit from the cell cycle to G0 in normal (untransformed) cells. Upon restimulation with mitogenic signals, the cell returns to the same point in G1-pm from which it left the cell cycle. Thus the cell seems to have a memory for how far it has advanced through G1-pm, suggesting that a continuous structural alteration, for example chromatin decondensation, takes place in G1. The molecular background to transition from growth factor dependence in G1-pm to growth factor independence in G1-ps (a switch which represents commitment to a new cell cycle and passage through R) is still not fully understood. Cyclin-dependent kinase (cdk)-mediated hyperphosphorylation of the retinoblastoma protein (Rb), and concomitant liberation (and activation) of members of the E2F family of transcription factors, are probably important aspects of R control in normal cells. A key component here could be cdk2 activity which is controlled by cyclin E. When cdk2 activity starts to increase rapidly in G1, due to activation of a positive feedback loop, it reaches a critical level above which cdk inhibitors (CKIs) such as p21 and p27 are outweighed; the cell has then become independent of mitogenic and inhibitory signals and is committed to a new cell cycle. However, other components are probably also involved in R control. For instance, a 'cryptic' R (a G1-pm-like state) can be induced even in tumour cells that do not respond to growth factor starvation or protein synthesis inhibitors, and are therefore probably defective in the cdk-Rb-E2F pathway. Possibly, a certain degree of chromatin decondensation has to take place after mitosis in order to allow transcription of, for example, the cyclin E gene or other critical E2F targets. Although the molecular basis for restriction point control still remains unclear, we can expect rapid progress in this important field over the next few years.

Comparative genomic hybridization of formalin-fixed, paraffin-embedded breast tumors reveals different patterns of chromosomal gains and losses in fibroadenomas and diploid and aneuploid carcinomas.

Comparative genomic hybridization serves as a screening test for regions of copy number changes in tumor genomes. We have applied the technique to map DNA gains and losses in 33 cases of formalin-fixed, paraffin-embedded primary breast tumors (13 fibroadenomas and 10 diploid and 10 aneuploid carcinomas). No genomic imbalances were found in fibroadenomas. Recurrent findings in adenocarcinomas include copy number increases for chromosomes 1q (14 of 20 samples), 8q (10 of 20), 17q (5 of 20), 6p (3 of 20), 13q (3 of 20), and 16p (3 of 20), and copy number decreases for chromosomes 22 (7 of 20), 17p (6 of 20), and 20 (3 of 20). Regional high level copy number increases were observed on chromosome bands 1q32, 8p11, 8q24, 10p, 11q13, 12p, 12q15, 17q11-12, and 17q22-24. The majority of the samples were studied for gene amplification of c-myc, c-erbB2, cycD1, and int-2 by means of Southern blot analysis. The comparison with DNA ploidy measurements revealed a different distribution and a significantly higher number of chromosomal aberrations in aneuploid tumors than in diploid tumors and in fibroadenomas.

Absence of mutation at the GAP-related domain of the neurofibromatosis type 1 gene in sporadic neurofibrosarcomas and other bone and soft tissue sarcomas.

The NF1 gene encodes neurofibromin, a GTPase-activating protein containing a GAP-related domain (NF1-GRD) that is capable of downregulating ras by stimulating ras intrinsic GTPase activity. We tested 44 sarcomas, nine of which corresponded to sporadic neurofibrosarcomas, for mutations at the NF1-GRD by the polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) technique, finding no mutation in every sample tested. We suggest that inactivation of the NF1-GRD by gene mutation seems not to be an important event in the tumorigenesis of sarcomas.