Distinctive patterns of Her-2/neu, c-myc, and cyclin D1 gene amplification by fluorescence in situ hybridization in primary human breast cancers.

BACKGROUND: Human solid tumors undergo clonal evolution as they progress, but evidence for specific sequences of genetic changes that occur in individual tumors and are recapitulated in other tumors is difficult to obtain. METHODS: Patterns of amplification of Her-2/neu, c-myc, and cyclin D1 were determined by fluorescence in situ hybridization (FISH) in relation to the presence of p53 dysfunction and ploidy in 60 primary human breast cancers. RESULTS: We show that there are clusters of genophenotypic abnormalities that distinguish lobular breast cancers from nonlobular tumors; that cyclin D1 amplification occurs prior to the divergence of lobular breast cancers from nonlobular cancers; that p53 dysfunction, Her-2/neu amplification, and c-myc amplification are characteristic features of nonlobular breast cancers, but not of lobular breast cancers; and that the frequencies of amplification of all three oncogenes examined increase progressively with increasing aneuploidy, but that each gene exhibits a different profile of increasing amplification in relation to tumor progression. Early amplification of c-myc appears to be an especially prominent feature of hypertetraploid/hypertetrasomic tumors. CONCLUSIONS: The data suggest that in tumors containing multiple abnormalities, these abnormalities often accumulate in the same cells within each tumor. Furthermore, the same patterns of accumulation of multiple genophenotypic abnormalities are recapitulated in different tumors.

Correlations among p53, Her-2/neu, and ras overexpression and aneuploidy by multiparameter flow cytometry in human breast cancer: evidence for a common phenotypic evolutionary pattern in infiltrating ductal carcinomas.

Human solid tumors develop multiple genetic abnormalities that accumulate progressively in individual cells during the course of tumor evolution. We sought to determine whether there are specific sequences of occurrence of these progressive evolutionary changes in human breast cancers by performing correlated cell-by-cell measurements of cell DNA content, p53 protein, Her-2/neu protein, and ras protein by multiparameter flow cytometry in 56 primary tumor samples obtained at surgery. In addition, p53 allelic loss and Her-2/neu gene amplification were determined by fluorescence in situ hybridization in cells from the same samples. We reasoned that if there is a specific order in which genetic changes occur, the same early changes would be found consistently in the cells with the fewest abnormalities. We reasoned further that late-developing abnormalities would not occur alone in individual cells but would almost always be found together with the early changes inherited by the same cells. By these criteria, abnormalities involving p53 generally occurred early in the course of development of invasive breast cancers, whereas ras protein overexpression was found to be a late-occurring phenomenon. Within individual tumors, cellular p53 overexpression was often observed alone in individual cells, whereas ras protein overexpression was rarely observed in the absence of p53 overexpression and/or Her-2/neu overexpression in the same cells. Furthermore, the intracellular level of each abnormally expressed protein was found to increase progressively as new abnormalities were acquired. Infiltrating ductal carcinomas exhibited characteristic phenotypic patterns in which p53 allelic loss and/or p53 protein overexpression, Her-2/neu amplification and/or overexpression, aneuploidy, and ras overexpression accumulated within individual cells. However, this pattern was not a prominent feature of lobular breast cancers. All six lobular breast cancers studied were diploid. p53 allelic loss and/or early p53 overexpression, and late ras cooverexpression in the same cells were less common in lobular breast cancers than in infiltrating ductal carcinomas. Although Her-21neu overexpression was a common finding in lobular breast cancers, Her-2/neu amplification was not observed in these tumors.

Intracellular coexpression of epidermal growth factor receptor, Her-2/neu, and p21ras in human breast cancers: evidence for the existence of distinctive patterns of genetic evolution that are common to tumors from different patients.

Multiparameter flow cytometry studies were performed on cells from the primary tumors of 94 patients with breast cancer. Correlated cellular measurements of cell DNA content, Her-2/neu, epidermal growth factor receptor (EGFR), and p21ras levels were performed on each of 5,000 to 100,000 cells from each tumor. When criteria for positivity were matched with those in common use for immunohistochemical studies, 28 of 94 (30%) breast cancers were classified as positive for Her-2/neu overexpression. When similar criteria were applied to the EGFR measurements, 23 of 94 (24%) cases were classified as positive for EGFR overexpression. Similarly, 23 of 94 (24%) cases were classified as positive for p21ras overexpression. By conventional flow cytometric criteria for DNA ploidy, 24 cases were diploid, 28 were tetraploid, and 42 were aneuploid. When the measurements were treated as separate sets of data, the only statistically significant correlations noted were the high frequency of diploid tumors, which did not overexpress any of the three oncogenes studied (P < 0.05), and an association between Her-2/neu overexpression and aneuploidy (P < 0.03). When the data were treated as correlated intracellular measurements, 90 of the 94 tumors studied contained a population of cells in which the intracellular levels of Her-2/neu expression were directly correlated with the levels of EGFR expression in the same cells. The ratio of Her-2/neu molecules to EGFR molecules in the same cells exceeded 1 in the majority of tetraploid and aneuploid cases and was close to or less than 1 in the majority of diploid cases. In nearly all tumors, p21ras overexpression was observed only in cells that overexpressed Her-2/neu, EGFR, or both, and p21ras levels per cell were more closely correlated with levels of EGFR per cell in the same cells than with Her-2/neu levels per cell. The data are consistent with a model in which heterodimerization of Her-2/neu and EGFR in individual cells is achieved by one of several genetic evolutionary pathways, all of which commonly lead to p21ras overexpression. The two major genetic evolutionary pathways identified in this study are an aneuploid, Her-2/neu overexpression-driven pathway seen in 59 of 94 tumors, and a diploid, EGFR overexpression-driven pathway seen in 19 of 94 tumors. All tumors with Her-2/neu:EGFR ratios greater than 2 contained an infiltrating ductal carcinoma component, whereas all infiltrating pure lobular carcinomas had Her-2/ neu:EGFR ratios that were less than 2. All of the genetic evolutionary pathways identified in this study were represented among the 11 tumors from patients who experienced early tumor recurrences.

The accumulation of multiple genetic abnormalities in individual tumor cells in human breast cancers: clinical prognostic implications.

PURPOSE: Human solid tumors undergo multiple genetic evolutionary changes as they evolve from the normal state to advanced stages of malignancy. This study characterizes the degree of advancement of primary human breast cancers in their genetic evolutionary pathways, and determines if this is of clinical significance. MATERIALS AND METHODS: Correlated cell-by-cell measurements of cell DNA content, HER-2/neu protein content per cell, and H-ras protein content per cell were obtained by means of multiparameter flow cytometry on primary tumors from 95 patients with clinically localized breast cancer. Laboratory findings were correlated with subsequent clinical course in 91 of these patients. RESULTS: Multiple genetic abnormalities were found to accumulate in individual cells in primary human breast cancers. Almost all tumors contained subsets of cells with one, two, or three abnormalities per cell in various combinations. After a median follow-up time of 32 months, 11 of 13 patients with early recurrence had primary tumors in which more than 5% of cells were hypertetraploid, overexpressed HER-2/neu protein, and overexpressed H-ras protein (triple-positive cells). The duration of disease-free survival among patients with primary tumors that contained triple-positive cells was significantly shorter than for patients whose tumors did not contain triple-positive cells. The presence of subpopulations of cells with maximums of only one abnormality per cell or only two abnormalities per cell, in any combination, was of no prognostic significance. Among patients whose nodal status was known, 12 had recurrent disease, and all had positive axillary nodes. Among 36 patients known to have negative axillary nodes, no recurrence has been reported to date. CONCLUSIONS: The number of genetic abnormalities that accumulate in individual cells in primary breast cancers reflects the degree of advancement of a tumor in its genetic evolutionary sequence, and provides useful clinical prognostic information. Because follow-up duration is still relatively short, and because disease in node-negative patients tends to recur later than in node-positive patients, it is still too early to know if three measurements per cell will be sufficient to improve prognosis in node-negative disease.

Assessing sequential oncogene amplification in human breast cancer.

Studies of amplification and/or overexpression of c-myc, HER-2/neu, and H-ras in breast cancer have shown that each is associated with a poor prognosis. The purpose of this study was to explore the possibility that there is a preferred sequence of amplification of these oncogenes in breast cancer. The frequencies of amplification and patterns of co-amplification of c-myc, HER-2/neu, and H-ras were studied in a group of 84 breast cancers. The data suggested a preferred sequence of amplification that consisted of c-myc amplification-HER-2/neu amplification-H-ras amplification. This model was supported by loglinear analysis. In addition, the levels of amplification of JC-A, a DNA fragment newly isolated from a patient with advanced breast cancer, were studied in 61 of these cases. The data suggested that JC-A amplification occurred early. Loglinear analysis supported a model in which JC-A amplification occurred either before or after c-myc amplification but was unrelated to Her-2/neu or ras amplification.

Preferred genetic evolutionary sequences in human breast cancer: a case study.

Multiparameter flow cytometry studies were performed on the cells of an aggressive human breast cancer at the time of diagnosis and at relapse. The aneuploid cells that overexpressed large amounts of both HER-2/neu and ras survived intensive chemotherapy and were responsible for tumor relapse. At relapse, these cells were shown to overexpress simultaneously at least five oncogenes: HER-2/neu, ras, EGF receptor, p53 and c-myc. A partial reconstruction of the genetic evolutionary sequence in this tumor indicated that HER-2/neu overexpression was an early step in the sequence. Subsequent HER-2/neu overexpression, EGF receptor overexpression and p53 protein overexpression were each associated with ras overexpression. The data suggest that ploidy and oncogene overexpression cannot be used as independent clinical prognostic factors. The ability to characterize tumors according to the degree of advancement in the genetic evolutionary might serve as a basis for genetic staging for adjuvant therapy.

Sequential paraformaldehyde and methanol fixation for simultaneous flow cytometric analysis of DNA, cell surface proteins, and intracellular proteins.

A cell fixation and permeabilization procedure consisting of sequential paraformaldehyde and methanol was evaluated and found suitable for concomitant flow cytometric quantification of total cellular DNA, immunofluorescence measurements of cell surface proteins, and immunofluorescence measurements of intracellular proteins. Paraformaldehyde/methanol-fixed cells exhibited significantly greater intracellular antitubulin immunofluorescence than cells fixed with paraformaldehyde or methanol alone (p less than 0.002) and significantly greater intracellular antitubulin immunofluorescence than cells fixed with methanol followed by paraformaldehyde (p less than 0.006). With paraformaldehyde/methanol fixation, cell morphology was well preserved and forward and right angle light scatter properties were sufficiently well maintained to permit gating on these parameters. Cell surface marker staining with fluorescent anti-leukocyte antibodies was unaffected by fixation with paraformaldehyde/methanol. Paraformaldehyde effects on the intensity of DNA staining with propidium iodide were dependent on paraformaldehyde concentration and fixation temperature; these effects were least pronounced at low paraformaldehyde concentrations (0.25% or less), and at temperatures lower than 37 degrees C. Paraformaldehyde fixation may result in differences in propidium iodide staining of DNA in some diploid cells, which may produce small spurious aneuploid peaks in normal peripheral blood leukocytes. Paraformaldehyde fixation also produces an apparent increase in the DNA index of aneuploid cell populations in comparison with methanol fixation, particularly when the DNA index exceeds 1.5. Occasionally, this paraformaldehyde fixation-induced effect is useful in identifying biologically distinct near-diploid subpopulations in tumors.

Total translation of vasopressin and oxytocin in neurohypophysis of rats.

There is a paucity of information about total translation rate of vasopressin and oxytocin. Because the site of synthesis of the neurohypophysial hormones is anatomically separate from the site of storage, we were able to measure total translation by blocking transport of newly synthesized hormone and measuring accumulation in the areas of synthesis in the hypothalamus. Colchicine administered into the third ventricle in doses as low as 3.5 micrograms/rat blocked transport for 18 h. The linear increase in vasopressin and oxytocin content over 18 h indicated a stable rate of synthesis, which was 1.2 and 1.9 pmol/h for vasopressin and 1.4-2.5 pmol/h for oxytocin. The molar correlation for synthesis of total neurophysin to total hormone was 1.16. Infusion of oxytocin and vasopressin into rats indicated that this level of synthesis of hormone was essential under steady-state conditions to maintain plasma levels in the low physiological range of approximately 3 pg/ml for oxytocin and 1 pg/ml for vasopressin. The data on total synthesis of the neurohypophysial hormones provide a reference for studies in which physiological replacement is required and also provide the technique and base-line data to determine how translation of vasopressin and oxytocin is regulated when neurohypophysial function is altered.

cis-acting elements of the rat growth hormone gene which mediate basal and regulated expression by thyroid hormone.

In GC cells, a growth hormone-producing rat pituitary cell line, 3,5,3'-triiodo-L-thyronine (L-T3) rapidly stimulates the transcription rate of the growth hormone gene which parallels the level of chromatin-associated L-T3-receptor complexes (Yaffe, B. M., and Samuels, H. H. (1984) J. Biol. Chem. 259, 6284-6291). In this study we have functionally mapped the elements of the gene which are involved in mediating basal and hormone-regulated expression. Stable transformation studies indicate that transcriptional regulation of the gene by L-T3 is mediated by sequences in the 5'-flanking region. Transient expression studies were performed using a series of chimeric plasmids in which 5'-flanking DNA was ligated to the chloramphenicol acetyltransferase gene. Transient expression occurred only in cells which expressed the endogenous growth hormone gene. Sequences between -104 and +7 were found to be essential for basal expression. One of the most highly conserved regions (-105 to -145) contains elements which further enhance the level of basal expression but are not necessary for regulated expression by L-T3. DNA between -210 and -181 was found to be essential for stimulation by L-T3 and was shown to function most efficiently with the homologous rat growth hormone promoter (-104 to +7). Sequences from -206 to -198 show about 80% homology with a sequence in the 5'-flanking region of two other rat genes which are regulated by thyroid hormone. Glucocorticoid hormones, which also transcriptionally stimulate the rat growth hormone gene, elicited only minimal effects in both stable and transient expression studies. This suggests that the elements which mediate glucocorticoid regulation of the endogenous gene are found either upstream of the cloned 5'-flanking region (1800 base pairs) or 3' of the cap site.

Prolonged hypothermia after phenobarbital-induced anesthesia in adrenalectomized rats.

Adrenalectomized rats are hypersensitive to the hypothermic as well as hypnotic effects of phenobarbital. The exaggerated hypnotic response is more pronounced in the chronic (10 day) than the acute (1 day) adrenalectomized rat and is reversed by glucocorticoid replacement. The prolonged hypothermia to a hypnotic dose of phenobarbital occurs in adrenalectomized but not adrenodemedullated rats. This hypersensitivity is characterized by an impaired ability to regain normal body temperatures for as long as 24 hours after regaining the righting reflex. The prolonged hypothermia is prevented by prior treatment with glucocorticoids but not mineralocorticoids. There appears to be no gross alteration in disposition of phenobarbital following adrenalectomy suggesting that the prolonged duration of hypothermia is not a consequence of accumulation of the drug.

Radiochemical evidence for estradiol-17-fatty acid esters in human blood.

The C-17 fatty acid esters of estradiol, known as the lipoidal derivatives of estradiol, LE2, are metabolites of estradiol that were originally isolated from various tissues after in vitro incubations with estradiol. These steroidal esters are active estrogens with extremely prolonged potencies. The present study investigates the existence of LE2 in human blood using a radiochemical isotope dilution technique. The LE2 fraction from blood was isolated, saponified, and the hydrolyzed estradiol was then acetylated with [3H]acetic anhydride. It was found that 3H was incorporated into estradiol diacetate, demonstrating that LE2 is present in human blood. Thus these steroidal esters represent a new class of endogenous estrogens which have not been previously considered in the physiology of the female sex steroids.

5'-Flanking DNA of the rat growth hormone gene mediates regulated expression by thyroid hormone.

Thyroid hormone has been shown to rapidly stimulate the rate of rat growth hormone gene transcription which parallels the kinetics of binding of 3,5,3'-triiodo-L-thyronine (L-T3) to its nuclear receptor (Yaffe, B. M., and Samuels, H. H. (1984) J. Biol. Chem. 259, 6284-6291). We have constructed a chimeric gene to explore whether the 5'-flanking region of the rat growth hormone gene contains a DNA element which could mediate thyroid hormone control of growth hormone gene expression. The construct consists of 1.8 kilobase pairs of the 5'-flanking region extending 11 nucleotides downstream from the transcription initiation (cap) site ligated to Escherichia coli DNA containing the structural gene for the enzyme xanthine-guanine phosphoribosyltransferase. GC cells, a growth hormone producing rat pituitary cell line, were transfected with this chimeric gene and stable transformants in which the enzyme is regulated by L-T3 were isolated by positive selection using mycophenolic acid and xanthine. These stable transformants develop with relatively high frequency and show marked L-T3 stimulation of xanthine-guanine phosphoribosyltransferase mRNA which is initiated at the cap site of the growth hormone gene. This study provides the first evidence that the 5'-flanking region of the rat growth hormone gene contains a DNA regulatory element which can mediate control of gene expression by thyroid hormone.

The interaction of C-17 esters of estradiol with the estrogen receptor.

Various C-17 alkyl esters of estradiol (E2) were tested as ligands for the estrogen receptor. The naturally occurring fatty acid esters, represented by E2-17-stearate, E2-17-palmitate, and E2-17-arachidonate, did not compete with [3H]E2 for receptor-binding sites. [3H]E2-17-stearate did not bind in a specific or saturable manner to uterine cytosol. On the other hand, the long-acting pharmacological esters of E2, E2-17-acetate, -propionate, -valerate, etc., were highly effective in competing for the binding of [3H]E2 to the uterine receptor. However, it was found that these short chain esters were hydrolyzed to E2 when incubated with uterine cytosol under the conditions used for the binding assay. Consequently, [3H]E2-17-acetate and [3H] E2-17-valerate were tested as ligands in an assay in which the hydrolytic activity was minimized by partially purifying the receptor with (NH4)2SO4 precipitation and by limiting the duration of the incubation. In this assay there was no specific binding of the C-17-valerate ester. There was a relatively small amount of specifically bound radioactivity in the incubation of the acetate ester, but upon high performance liquid chromatographic analysis, the specifically bound steroid was found to be E2 and not the ester. These results indicate that the C-17 esters of E2, from C2 to the long chain fatty acids, all of which are potent long-acting estrogens, exert their estrogenic effects only after hydrolysis to the free C18 steroid.

Estradiol fatty acid esters occur naturally in human blood.

Treatment of nonpolar ether extracts of human female blood with mild alkali produced more immunoassayable estradiol than the unhydrolyzed extract. Analysis of the serum extracts showed that the substance which released immunoreactive estradiol after hydrolysis has chromatographic properties identical to those of fatty acid esters of estradiol esterified at carbon 17. The physiological role of these previously unknown endogenous esters might be inferred from their structural similarity to synthetic drugs used therapeutically for their prolonged estrogenic action.