Prognostic and predictive role of ESR1 status for postmenopausal patients with endocrine-responsive early breast cancer in the Danish cohort of the BIG 1-98 trial.

BACKGROUND: Estrogen Receptor 1 (ESR1) aberrations may be associated with expression of estrogen receptor (ER) or progesterone receptor (PgR), human epidermal growth factor receptor-2 (HER2) or Ki-67 labeling index and prognosis. PATIENTS AND METHODS: ESR1 was assessed in 1129 (81%) of 1396 postmenopausal Danish women with early breast cancer randomly assigned to receive 5 years of letrozole, tamoxifen or a sequence of these agents in the Breast International Group 1-98 trial and who had ER ≥ 1% after central review. RESULTS: By FISH, 13.6% of patients had an ESR1-to-Centromere-6 (CEN-6) ratio ≥ 2 (amplified), and 4.2% had ESR1-to-CEN-6 ratio <0.8 (deleted). Deletion of ESR1 was associated with significantly lower levels of ER (P < 0.0001) and PgR (P = 0.02) and more frequent HER2 amplification. ESR1 deletion or amplification was associated with higher-Ki-67 than ESR1-normal tumors. Overall, there was no evidence of heterogeneity of disease-free survival (DFS) or in treatment effect according to ESR1 status. However, significant differences in DFS were observed for subsets based on a combination of ESR1 and HER2 status (P = 0.02). CONCLUSIONS: ESR1 aberrations were associated with HER2 status, Ki-67 labeling index and ER and PgR levels. When combined with HER2, ESR1 may be prognostic but should not be used for endocrine treatment selection in postmenopausal women with endocrine-responsive early breast cancer.

Frequent amplifications and deletions of G1/S-phase transition genes, CCND1 and MYC in early breast cancers: a potential role in G1/S escape.

Uncontrolled growth of cancer cells can be related to dysfunctional cell cycle control, including entry into S-phase, initiating cell division. Cyclin CCND3 and CCNE1 along with CDK2 and CDK6 regulate this checkpoint, and genetic changes, detectable by fluorescence in situ hybridization, are hypothesized to increase the aggressiveness of breast cancer, thereby influencing patient survival. Genomic change was investigated in 106 primary breast cancer samples, where the combined gene copy number changes in one of these four cell cycle regulatory factors was observed in 22% of the 98 tumors of successful analysis, distributed with 15 deletions and 7 amplifications. A trend towards decreased survival was observed with the aberrations, suggesting a prognostic potential of this set of markers, which was supported by an association with tumor grade. For validation of the new set of FISH probes for the G1/S-phase cell cycle factors, two additional markers, frequently amplified in breast cancers, were included in this study: The G1/S phase control gene CCND1 and the proliferation marker MYC. Both markers were amplified in 14% and deleted in 5% of the cases. This is the first report of genomic deletions of MYC in breast cancer.

Is PTEN loss associated with clinical outcome measures in human prostate cancer?

Inactivating PTEN mutations are commonly found in prostate cancer, resulting in an increased activation of Akt. In this study, we investigate the role of PTEN deletion and protein expression in the development of hormone-refractory prostate cancer using matched hormone-sensitive and hormone-refractory tumours. Fluorescent in situ hybridisation and immunohistochemistry was carried out to investigate PTEN gene deletion and PTEN protein expression in the transition from hormone-sensitive to hormone-refractory prostate cancer utilising 68 matched hormone sensitive and hormone-refractory tumour pairs (one before and one after hormone relapse). Heterogeneous PTEN gene deletion was observed in 23% of hormone sensitive tumours. This increased significantly to 52% in hormone-refractory tumours (P=0.044). PTEN protein expression was observed in the membrane, cytoplasm and the nucleus. In hormone sensitive tumours, low levels of cytoplasmic PTEN was independently associated with shorter time to relapse compared to high levels of PTEN (P=0.028, hazard ratio 0.51 (95%CI 0.27-0.93). Loss of PTEN expression in the nucleus of hormone sensitive tumours was independently associated with disease-specific survival (P=0.031, hazard ratio 0.52, 95%CI 0.29-0.95). The results from this study demonstrate a role for both cytoplasmic and nuclear PTEN in progression of prostate cancer to the hormone-refractory state.

Genetic alterations of CCND1 and EMSY in breast cancers.

AIMS: CCND1 and EMSY, on 11q13, are frequently amplified in breast cancer. CCND1 is implicated in cell cycle progression and EMSY is a BRCA2-associated repressor protein. The aim was to investigate gene copy numbers of CCND1 and EMSY and to determine if CCND1 amplification is associated with reduced survival of tamoxifen-treated breast cancer patients. METHODS AND RESULTS: Fluorescence in situ hybridization (FISH) was performed on 111 consecutive and 354 oestrogen receptor (ER)+ tamoxifen-treated breast cancers. In the consecutive set, CCND1 and EMSY were amplified in 14.8% and 7.2%, respectively, and deleted in 8.7% and 13.5%, respectively. In the ER+ set, CCND1 and EMSY were amplified in 20.6% and 9.6%, respectively, and deleted in 1.7% and 4.2%, respectively. CCND1 and EMSY gene amplifications were associated with decreased overall survival (OS) (P = 0.03 and P = 0.04, respectively) of patients in the ER+ set. CONCLUSION: As hypothesized, CCND1 amplifications are associated with poor OS in ER+ patients. EMSY amplification is also associated with poor OS. However, as >70% of EMSY amplifications were CCND1 amplified, EMSY may not have any additional effect on survival of ER+ breast cancer.

Sex-specific telomere length profiles and age-dependent erosion dynamics of individual chromosome arms in humans.

During aging, telomeres are gradually shortened, eventually leading to cellular senescence. By T/C-FISH (telomere/centromere-FISH), we investigated human telomere length differences on single chromosome arms of 205 individuals in different age groups and sexes. For all chromosome arms, we found a linear correlation between telomere length and donor age. Generally, males had shorter telomeres and higher attrition rates. Every chromosome arm had its individual age-specific telomere length and erosion pattern, resulting in an unexpected heterogeneity in chromosome-specific regression lines. This differential erosion pattern, however, does not seem to be accidental, since we found a correlation between average telomere length of single chromosome arms in newborns and their annual attrition rate. Apart from the above-mentioned sex-specific discrepancies, chromosome arm-specific telomere lengths were strikingly similar in men and women. This implies a mechanism that arm specifically regulates the telomere length independent of gender, thus leading to interchromosomal telomere variations.

BRCA1 and BRCA2 mutation status and cancer family history of Danish women affected with multifocal or bilateral breast cancer at a young age.

INTRODUCTION: A small fraction of breast cancer is the result of germline mutations in the BRCA1 and BRCA2 cancer susceptibility genes. Mutation carriers frequently have a positive family history of breast and ovarian cancer, are often diagnosed at a young age, and may have a higher incidence of double or multiple primary breast tumours than breast cancer patients in general. OBJECTIVES: To estimate the prevalence and spectrum of BRCA1 and BRCA2 mutations in young Danish patients affected with bilateral or multifocal breast cancer and to determine the relationship of mutation status to family history of cancer. SUBJECTS: From the files of the Danish Breast Cancer Cooperative Group (DBCG), we selected 119 breast cancer patients diagnosed before the age of 46 years with either bilateral (n=59) or multifocal (n=61) disease. METHODS: DNA from the subjects was screened for BRCA1 and BRCA2 mutations using single strand conformation analysis (SSCA) and the protein truncation test (PTT). Observed and expected cancer incidence in first degree relatives of the patients was estimated using data from the Danish Cancer Registry. RESULTS: Twenty four mutation carriers were identified (20%), of whom 13 had a BRCA1 mutation and 11 carried a BRCA2 mutation. Two mutations in BRCA1 were found repeatedly in the material and accounted for seven of the 24 (29%) mutation carriers. The mutation frequency was about equal in patients with bilateral (22%) and multifocal breast cancer (18%). The incidence of breast and ovarian cancer was greatly increased in first degree relatives of BRCA1 and BRCA2 mutation carriers, but to a much lesser degree in relatives of non-carriers. An increased risk of cancer was also noted in brothers of non-carriers. CONCLUSIONS: A relatively broad spectrum of germline mutations was observed in BRCA1 and BRCA2 and most of the mutations are present in other populations. Our results indicate that a diagnosis of bilateral and multifocal breast cancer is predictive of BRCA1 and BRCA2 mutation status, particularly when combined with information on the patients' age at diagnosis and family history of breast/ovarian cancer.

Telomere erosion varies during in vitro aging of normal human fibroblasts from young and adult donors.

The life span of normal fibroblasts in vitro (Hayflick limit) depends on donor age, and telomere shortening has been proposed as a potential mechanism. By quantitative fluorescence in situ hybridization and Southern blot analysis, we show progressive telomere loss to about 5 kb mean telomere restriction fragment length in fibroblasts from two adult donors within 40 population doublings, whereas in fibroblasts from two infant donors, telomere erosion is reduced, leaving a mean telomere restriction fragment length of approximately 7 kb at senescence (after approximately 60 population doublings). Aging of fibroblasts from both infant and adult donors was not accompanied by chromosomal abnormalities but was correlated with increased telomere repeat-binding factor 2 expression at both the protein and transcriptional level.

Complete loss of wild-type TP53 in a nontransformed human epithelial cell line is preceded by a phase during which a heterozygous TP53 mutant effectively outgrows the homozygous wild-type cells.

HMT-3522 is a spontaneously immortalized cell line derived from a fibrocystic breast lesion. After continuous accumulation of genetic changes, the cell line was transformed from a nontumorigenic to a malignant phenotype. One of the earliest genetic aberrations is a missense mutation of codon 179 (His179Asn) in the tumor suppressor gene TP53 leading to outgrowth of a cell type expressing only the mutant form of TP53. In this report, we extend earlier investigations to reveal the genetic background for the evolution from homozygous wild type to hemizygous mutated cells. The status of the TP53 alleles was followed at different stages by fluorescence in situ hybridization (FISH) and allele-specific PCR (ASPCR) on total DNA, as well as flow-sorted chromosomes--taking advantage of a size difference between the two homologues of chromosome 17 that harbor TP53 on 17p. This further allowed us to determine on which of the two chromosomes the mutated allele was located. The results presented here show that the cells have undergone an evolution from homozygous wild type for TP53 to heterozygous (His179Asn mutation in one allele), and finally to a hemizygous mutated state (deletion of the remaining wild-type allele). The finding of a transient period in which heterozygous cells dominate the population before the eventual outgrowth of hemizygous cells strongly indicates that the His179Asn mutation results in a tp53 protein with a dominant negative effect that does not totally abrogate the function of wild type TP53 in vitro.

Mink 5S rRNA genes map to 2q in three loci suggesting conservation of synteny with human 1q.

By in situ hybridization we show that the SS rRNA genes in the mink map to chromosome 2q in three loci. The 2q1.1 locus containing 34% of the 5S rDNA, maps close to the centromere, and the remaining two loci of the 5S rDNA map to 2q1.3 (52%) and to 2q2.3. (14%). These data were obtained with a tritiated transcript of the 5S rRNA gene containing 121 bp. In a comparative FISH study performed with a biotinylated transcript of the 5S rRNA gene the procedure failed to detect the 2q2.3 site. A closely corresponding difference between the two procedures experienced previously in man and in the crab-eating macaque is discussed. The present results suggest a homology between 2q in the mink and part of 1q in man harbouring the 5S rRNA genes in 1q42.13 and 1q31, respectively.

Molecular cytogenetic analysis of a nontumorigenic human breast epithelial cell line that eventually turns tumorigenic: validation of an analytical approach combining karyotyping, comparative genomic hybridization, chromosome painting, and single-locus fluorescence in situ hybridization.

The immortalized, nontumorigenic human breast epithelial cell line HMT-3522 has been used as a model for premalignant and, eventually, malignant development. During cultivation, the karyotype evolution was followed. At an early stage, a very long constant phase showed a near-diploid karyotype, with only five marker chromosomes. DNA from this phase was used for comparative genomic hybridization (CGH) analysis, confirming a previously known MYC amplification, and the integration sites were subsequently determined by single-locus fluorescence in situ hybridization (FISH). Furthermore, gains of 5q22-qter and 20q11-qter and deletion of most of chromosome 6 (6p23-qter) were detected by CGH. Because of uncertainty about some of the indicated changes, including a deletion of Ip35-pter, the CGH findings were investigated more closely by chromosome painting, leading to a revision of the karyotype: 45,XX,del(I)(p35),-6,dup(8)(pter-->qter::qter-->q24),der(12) t(6;12)(p23; p13),der(14)t(5;14)(q22;q32.3),der(17)t(8;17;20)(17pter-->17q25 ::8qter--> 8q23::8q24-->8qter::8q24-->8qter:: 8q23-->8q24.1::20q11-->20qter). Some karyotypic changes were confirmed by CGH; others had to be revised; and, in the Ip35 region, classical cytogenetics seems superior to CGH. However, CGH revealed a karyotypically unsuspected dup(20q) that might be of special relevance to breast tumor initiation or progression. Our study confirms that CGH is supplementary to current technologies, e.g., karyotyping and Southern analysis, but cannot replace them. In addition, our cell line turned out to be an excellent model for comparison among the different methods. The results imply that future cytogenetic analyses of complex karyotypes should be based on a combination of karyotyping, CGH, and FISH.

Trisomy 7p and malignant transformation of human breast epithelial cells following epidermal growth factor withdrawal.

We have reported previously on the first spontaneously immortalized, nonmalignant human breast epithelial cell line, HMT-3522, which is entirely dependent on exogenous epidermal growth factor (EGF). In passage 118, cells were adapted to grow in medium without EGF and a new growth-transformed subline, HMT-3522/gt-1, was generated and propagated at high growth rate without exogenous EGF (Madsen et al., Cancer Res., 52: 1210-1217, 1992). Here we have used this subline and the continuum of the parent line, HMT-3522/wt, to pose the question whether a relevant change in a physiological parameter of the microenvironment will induce malignant transformation. The two cell lines were cultured under identical conditions with the only exception that EGF was omitted in the medium for gt-1. Initially, wt and gt-1 were identical in terms of karyotype as well as morphology, growth rate, and protein expression as revealed by two-dimensional gel electrophoresis. A highly dramatic shift to phenotype was observed in passage 238 when the gt-1 line became tumorigenic in nude mice. After two mouse-culture passages, the resulting malignantly transformed cell line (HMT-3522/mt-1) was refractory to the growth-modulating effect of EGF and presented an extra copy of a chromosome marker, 7q-, as the only cytogenetic difference from the gt-1. Our results suggest that microenvironmental cues are powerful factors in the induction of malignancy. A major role of EGF receptor in the malignant transformation is emphasized by loss of EGF sensitivity and acquisition of an extra chromosome 7p harboring the EGF receptor gene. We hypothesize that during premalignant hyperplasia, a population of EGF/transforming growth factor alpha autonomous epithelial cells in situ may develop as a consequence of local transforming growth factor alpha deprivation, a condition reflected in the culture model as autonomy after EGF withdrawal.

Molecular definition of deletions of different segments of distal 5p that result in distinct phenotypic features.

Cri du chat syndrome (CDC) is a segmental aneusomy associated with deletions of chromosome 5p15. In an effort to define regions that produce the phenotypes associated with CDC, we have analyzed deletions from 17 patients. The majority of these patients had atypical CDC features or were asymptomatic. Using these patients, we have mapped several phenotypes associated with deletions of 5p, including speech delay, catlike cry, newborn facial dysmorphism, and adult facial dysmorphism. This phenotypic map should provide a framework with which to begin identification of genes associated with various phenotypic features associated with deletions of distal 5p. We have also analyzed the parental origin of the de novo deletions, to determine if genomic imprinting could be occurring in this region. In addition, we have isolated cosmids that could be useful for both prenatal and postnatal assessments of del5(p) individuals.

In vitro karyotype evolution and cytogenetic instability in the non-tumorigenic human breast epithelial cell line HMT-3522.

The "spontaneously" immortalized cell line HMT-3522, derived from a fibrocystic breast lesion, is used as a model for premalignant breast epithelium. During 205 passages the cytogenetic evolution was followed. The results were compared with our earlier results on oncogene expression and growth factor requirements. During in vitro growth, gain and loss of markers, loss of normal chromosomes, and duplication of the chromosome complement could be demonstrated. The variability increased during in vitro growth. This variability, probably created randomly, leads to cells with different growth capacities, from which sidelines may be selected and become stemlines. The karyotypic evolution (including polyploidization) demonstrated here may be a result of genetic instability and heterogeneity. Although tumorigenicity was not achieved, either due to lack of cancer-specific gene alterations or to lack of proper selection pressure, the results suggest an ongoing process towards malignancy.

Altered gene expression of c-myc, epidermal growth factor receptor, transforming growth factor-alpha, and c-erb-B2 in an immortalized human breast epithelial cell line, HMT-3522, is associated with decreased growth factor requirements.

Activation of protooncogenes and constitutive secretion of autocrine growth factors are thought to be involved in the uncontrolled growth of cancer cells. We have attempted to elucidate the role of oncogenes and growth factors in the premalignant progression of human breast epithelial cells by using an immortalized, nontumorigenic, near-diploid human mammary epithelial cell line, HMT-3522, derived from a fibrocystic lesion and established in our laboratory. During propagation in tissue culture, the growth factor requirements of the HMT-3522 cells decreased simultaneously with an amplification and overexpression of the c-myc protooncogene. Other protooncogenes related to human breast cancer were unaltered with regard to gene copy number and expression. In passage 118, in which the most important growth factor still was epidermal growth factor (EGF), we were able to isolate an EGF-independent subline (S2). The EGF independence of S2 was accompanied by an overexpression of the mRNAs for epidermal growth factor receptor (EGF-R), transforming growth factor-alpha, and c-erb-B2 as compared to the EGF-dependent subline (S1). Moreover, by application of a blocking anti-EGF-R antibody, growth of S2 cells in EGF-free medium was inhibited significantly, indicating that EGF-R was involved in an autocrine loop probably with transforming growth factor-alpha as ligand. Neither the late passages of S1 cells nor S2 cells were tumorigenic after subcutaneous transplantation to athymic mice. Our results indicate that c-myc amplification and overexpression are correlated with a decreased requirement for growth factors. Even when these alterations are combined with immortalization and EGF independence, they are insufficient for malignant transformation of these human breast epithelial cells.

Differential tumorigenicity of two autologous human breast carcinoma cell lines, HMT-3909S1 and HMT-3909S8, established in serum-free medium.

In a serum-free medium we have established two new human breast carcinoma cell lines from a single primary tumor. Cultures were maintained on chemically defined medium CDM3 or on minor modifications of this medium, Dulbecco's modified Eagle medium-Ham's F12 supplemented with epidermal growth factor, insulin, transferrin, estradiol, hydrocortisone, triiodothyronine, cyclic AMP, phosphoethanolamine, ethanolamine, fibronectin, fetuin, ascorbic acid, bovine serum albumin, and trace element salts including selenite (Petersen and van Deurs, Cancer Res., 47: 856-866, 1987). Primary cultures comprised both NADPH-neotetrazolium reductase-positive carcinoma cells and NADPH-neotetrazolium reductase-negative cells of stromal appearance, as well as normal epithelial cells (Petersen and van Deurs, Cancer Res., 46: 2013-2020, 1986). In subsequent passages the cells were monitored exclusively using the tumorigenicity assay on nude mice. Two cell lines, one nontumorigenic, HMT-3909S1, and one tumorigenic, HMT-3909S8, were selected from the primary cultures. Selection of S8 through subline S4 required transient supplementation of CDM3 with fetal calf serum. Permanent lines S1 and S8 were maintained on serum-free medium. Further characterization of the two cell lines in terms of normal breast gland differentiation (Petersen and van Deurs, Differentiation, 39: 197-215, 1988) was carried out using immunocytochemistry, immunochemistry, electron microscopy, and cytogenetics. S8 appeared to be identical with the NADPH-neotetrazolium reductase-positive carcinoma cells of the primary cultures, with a particular subpopulation of carcinoma cells in the tumor of origin, and with the tumorigenic cells of the nude mice. This subline was aneuploid, typically epithelial in morphology, and expressed keratins K8 and K18 and the glycoprotein MAM-6, typical of luminal epithelial cells in the normal breast gland. Subline S1 appeared more like the elongated cells in the primary cultures and like a second subpopulation of cells in the carcinoma of origin. However, S1 cells were in fact epithelial, since they expressed keratins. Also, S1 cells seemed to be a triploidation of a cell with close resemblance to S4, while only few cytogenetic differences were found between S4 and S8, suggesting an origin of S1 and S8 via S4 from a single hypothetical stem cell.

Cytogenetic analysis of in vitro karyotype evolution in a cell line established from nonmalignant human mammary epithelium.

Cytogenetic analysis was carried out on six passages (pass. 16, 19, 21, 25, 34, and 47) of the nontumorigenic epithelial cell line, HMT-3522, established from a fibrocystic lesion of the human breast. Minor chromosome abnormalities were present in the first passage (pass. 16) available for study, and limited cytogenetic progression was observed during the in vitro growth. A modal chromosome number of 45 chromosomes was found in all passages. Each passage contained 4-5 marker chromosomes. Three markers were consistently present in all passages studied. During in vitro growth two markers were gained and two markers were lost from the stemline karyotype. The two latest passages studied had identical karyotypes: 45,XX, del(1)(q44----p32:),t(5;14)(14p13----14q32::5q22----++ +5q35),t(6;8;12;17)(8p23---- 8q24::6p21.1----6p23;12q24----12p13::6p23- --- 6p25;17p13----17q25::6q11----6q27). The present study demonstrates chromosome abnormalities and karyotypic evolution in a nontumorigenic (in nude mice) and noninvasive (in vitro tested) cell line established from nonmalignant epithelial breast tissue. The results are discussed in relation to gene amplification, double minutes and oncogene localization.

Chromosome analysis of in situ breast cancer.

Eleven ductal carcinoma in situ (DCIS), 5 DCIS with microinvasion and 4 benign lesions have been investigated cytogenetically. Twelve of the 20 breast tumors (60%) had sufficient mitotic activity for chromosome analysis. All ductal carcinoma in situ had abnormal karyotypes, and clonal marker chromosomes could be identified in all tumors analyzed. None were cytogenetically normal. All but one of the 12 DCIS showed genetic heterogeneity. Tumor progression seems to be associated with loss of chromosomes.

Chromosome changes of in situ carcinomas in the female breast.

Eight lesions of ductal carcinoma in situ (DCIS) and one case of benign radial scar were investigated cytogenetically and compared with invasive ductal carcinomas. DCIS was shown to be associated with chromosome changes different from those of invasive carcinomas as concern ploidy levels. All cases exclusively contained abnormal metaphases, also the specimen that was classified as a benign radial scar. No consistent chromosome changes were found. Chromosome no. 1 was most frequently involved in marker chromosome formation.

DNA distribution and chromosome number in human cervical carcinoma.

The degree of ploidy of 15 human cervical carcinomas was investigated by use of flow cytometry and cytogenetic analysis. All tumors contained aneuploid cell populations. The results showed no correlation between ploidy and the clinical stage of the tumor. A high correlation was found between modal DNA content and modal chromosome number of the aneuploid cell populations, and it is concluded that flow cytometric analyses reflect the occurrence of tumor stemlines.