Comprehensive genomic profiling of epithelial ovarian cancer by next generation sequencing-based diagnostic assay reveals new routes to targeted therapies.

OBJECTIVE: Targeted next generation sequencing (NGS) was evaluated for its ability to identify unanticipated targetable genomic alterations (GA) for patients with relapsed ovarian epithelial carcinoma (OC). METHODS: DNA sequencing was performed for 3320 exons of 182 cancer-related genes and 37 introns of 14 genes frequently rearranged in cancer on indexed, adaptor ligated, hybridization-captured libraries using DNA isolated from FFPE sections from 48 histologically verified relapsed OC specimens. The original primary tumor was sequenced in 26 (54%) of the cases and recurrent/metastatic tumor site biopsies were sequenced in 22 (46%) of the cases. Actionability was defined as: GA that predict sensitivity or resistance to approved or standard therapies or are inclusion or exclusion criteria for specific experimental therapies in NCI registered clinical trials. RESULTS: There were 38 (80%) serous, 5 (10%) endometrioid, 3 (6%) clear cell, 1 mucinous (2%) and 1 (2%) undifferentiated carcinomas. 141 GA were identified with an average of 2.9 GA (range 0-8) per tumor, of which 67 were actionable for an average of 1.4 actionable GA per patient (range 0-5). 33/48 (69%) of OC patient samples harbored at least one actionable GA. Most common GA were TP53 (79%); MYC (25%); BRCA1/2 (23%); KRAS (16.6%) and NF1 (14.5%). One tumor featured an ERBB2 point mutation. One of 3 (33%) of clear cell tumors featured cMET amplification validated by both FISH and IHC. CONCLUSIONS: NGS assessment of therapy resistant OC identifies an unexpectedly high frequency of GA that could influence targeted therapy selection for the disease.

Estrogen inhibits the growth of estrogen receptor-negative, but not estrogen receptor-positive, human mammary epithelial cells expressing a recombinant estrogen receptor.

Estrogen is essential for the growth of the normal mammary gland and most estrogen receptor (ER)-positive mammary carcinomas. To better understand the differences between the estrogen response pathways in normal and tumor cells, we have stably transfected ER-negative immortal, nontumorigenic human mammary epithelial cells and ER-negative breast cancer cells with an ER-encoding expression vector. Unexpectedly, estrogen treatment (1.0 nM) inhibited the proliferation of ER-transfected nontumorigenic and tumor-derived cells. The control transfectants and parental cells exhibited no response to estrogen concentrations as high as 1.0 microM. This inhibitory effect was attributed to a decreased growth rate and a perturbation of the cell cycle distribution by estrogen treatment of the ER transfectants. The inhibitory response was blocked by cotreatment with the antiestrogen ICI 164,384 as predicted for a pure antagonist of estrogen action. However, treatment with the antiestrogen hydroxytamoxifen caused growth inhibition, implying that hydroxytamoxifen acts as an agonist of estrogen action in ER-transfected cells. Since estrogen is a mitogenic and not a growth-inhibitory stimulus for ER-positive breast cancers and cell lines, we tested the effect of constitutive, high level expression of the ER in ER-positive tumor cells. Stable transfection of ER-positive MCF-7 and T47D cells with the ER expression vector yielded cells with varying amounts of ER. At ER levels comparable to those found in the ER-negative transfected cells, the MCF-7 and T47D ER transfectants were not inhibited by estrogen. These data suggest that ER-positive breast cancer cells can tolerate higher constitutive levels of ER expression than ER-negative cells. The mechanism by which this is accomplished may be an essential step in the process which yields ER-positive tumors.

Expression of growth factors and oncogenes in normal and tumor-derived human mammary epithelial cells.

The expression of genes which may be involved in the regulation of human mammary epithelial cell growth [transforming growth factors alpha and beta] and tumorigenesis [c-myc, erbB2, epidermal growth factor receptor (EGFR), Ha-ras, pS2] has been compared in similarly cultured normal cell strains and tumor cell lines. We have found that the normal breast cells produce high levels of EGFR mRNA, which are translated into nearly 10(5) low affinity epidermal growth factor-binding molecules/cell. In the estrogen receptor-negative lines examined, the EGFR gene was expressed at levels comparable to those in the normal cells. In contrast, EGFR and transforming growth factor alpha mRNAs were reduced in estrogen receptor-positive tumor lines compared to estrogen receptor-negative lines and normal cells. Steady state mRNA levels for transforming growth factor beta, erbB2, c-myc, and Ha-ras in the normal cells were greater than or comparable to those in all of the breast tumor lines. Furthermore, in the absence of gene amplification, only one of the genes examined (i.e., pS2) was overexpressed in a subset of the tumor cells compared to their normal counterparts. Several reports by other investigators have described overexpression of some of these genes in breast biopsies and in tumor lines in studies lacking normal controls. Thus, our results, in which the same genes were not overexpressed compared to normal cells unless amplified, underscore the importance of including appropriate normal controls in studies aimed at defining aberrant patterns of gene expression in tumor cells.

Tumor progression in four mammary epithelial cell lines derived from the same patient.

Two primary and two metastatic cell lines with distinct phenotypes and genotypes have been established from a patient diagnosed as having infiltrating and intraductal mammary carcinoma (21T series). All four lines can be cultured in the same medium, DFCI-1, which also supports long-term growth of normal epithelial cells. Therefore, we have been able to compare normal and tumor cells at the cellular and molecular levels. The mammary origin of the 21T series was confirmed by using antibodies against the human milk fat globule antigen-2 epitope. The two primary tumor lines (21NT and 21PT) are both immortal and aneuploid, although only 21NT is tumorigenic in the nude mouse assay. The two populations derived from the metastatic pleural effusion (21MT-1 and 21MT-2) each exhibit distinct characteristics in morphology and growth factor requirements. The erbB2 gene is amplified and overexpressed in all of these cell lines compared to normal epithelial cell controls. These four tumor cell lines from a single patient represent a mammary tumor progression series that has been established in long-term cell culture.

Identification of gene expression profiles that predict the aggressive behavior of breast cancer cells.

With the goal of identifying genes that have an expression pattern that can facilitate the diagnosis of primary breast cancers (BCs) as well as the discovery of novel drug leads for BC treatment, we used cDNA hybridization arrays to analyze the gene expression profiles (GEPs) of nine weakly invasive and four highly invasive BC cell lines. Differences in gene expression between weakly and highly invasive BC cells were identified that enabled the definition of consensus GEPs for each invasive phenotype. To determine whether the consensus GEPs, comprising 24 genes, could be used to predict the aggressiveness of previously uncharacterized cells, gene expression levels and comparative invasive and migratory characteristics of nine additional human mammary epithelial cell strains/lines were determined. The results demonstrated that the GEP of a cell line is predictive of its invasive and migratory behavior, as manifest by the morphology of its colonies when cultured on a matrix of basement membrane constituents (i.e., Matrigel). We found that the expression of keratin 19 was consistently elevated in the less aggressive BC cell lines and that vimentin and fos-related antigen-1 (FRA-1) were consistently overexpressed in the more highly aggressive BC cells. Moreover, even without these three genes, the GEP of a cell line still accurately predicted the aggressiveness of the BC cell, indicating that the expression pattern of multiple genes may be used as BC prognosticators because single markers often fail to be predictive in clinical specimens.

Induction of estrogen-regulated genes differs in immortal and tumorigenic human mammary epithelial cells expressing a recombinant estrogen receptor.

Studies on estrogen receptor (ER)-positive human breast cancer cell lines have shown that estrogen treatment positively modulates the expression of the genes encoding transforming growth factor-alpha (TGF alpha), 52-kDa cathepsin-D, and pS2. To determine whether these genes would be similarly regulated by estrogens in normal human mammary epithelial cells, we stably transfected immortal nontumorigenic human mammary epithelial cells with an ER-encoding expression vector. ER-negative tumor cells were also transfected for comparison. Levels of TGF alpha and 52-kDa cathepsin-D mRNA were enhanced by estrogen treatment of both ER-transfected immortal and tumorigenic cells, demonstrating that the ER by itself is sufficient to elicit estrogenic regulation of the expression of these genes. In contrast, expression of the pS2 gene was detected only in the ER-transfected tumor cells. The ER in both cell lines is capable of recognizing the pS2 promoter, however, since estrogen enhanced the activity of an introduced pS2-CAT reporter plasmid in transient expression analyses. These and other experiments with somatic cell hybrids between the immortal cells and ER+/pS2+ MCF-7 tumor cells, where pS2 gene expression is extinguished, support the conclusion that the immortal nontumorigenic cells encode gene products that block endogenous pS2 expression. These results also imply that such repressors are not active in the tumor cells.

The novel estrogen-responsive B-box protein (EBBP) gene is tamoxifen-regulated in cells expressing an estrogen receptor DNA-binding domain mutant.

We have identified a 2.6-kb mRNA whose steady state levels are increased 2- to 4-fold by treatment of human mammary epithelial cells (HMEC) stably expressing an estrogen receptor (ER) transgene with either estrogen (E) or the antiestrogen, 4-hydroxy-tamoxifen (HT). The cDNA corresponding to this mRNA encodes a 564-amino acid protein, named estrogen-responsive B box protein (EBBP), that is a new member of a subfamily within the B box zinc finger protein family, which includes transcription factors (e.g. TIF1), tumor suppressor proteins (e.g. PML), and proteins implicated in development (e.g. ret finger protein, XNF7). The EBBP mRNA is detectable by Northern blot analysis in most tissues, with the exception of liver and peripheral blood lymphocytes, and the gene has been mapped to human chromosome 17p11.2. In contrast to most B box family members, EBBP has a predominantly cytoplasmic localization. Studies of the estrogenic regulation of EBBP expression demonstrated that the E-dependent increase in EBBP mRNA levels in the ER-transfected HMEC is an early, ER-mediated, and cycloheximide-insensitive process. In HMEC stably transfected with an ER mutant containing a deletion in the second zinc finger of the DNA-binding domain, E and HT had different effects on EBBP gene expression; EBBP regulation by E was dramatically reduced while the effects of HT were augmented. These data indicate that HT can modulate EBBP mRNA expression through a mutated ER, which has little activity when bound by E, and suggest that different molecular mechanisms control the E and HT responsiveness of the EBBP gene.

E1a inducibility of the adenoviral early E2a promoter is determined by specific combinations of sequence elements.

By transient expression analysis in HeLa cells of adenovirus-2 E2a early (E2aE) promoter mutants and hybrid E2aE-beta-globin gene constructs, we demonstrate the existence of three nucleotide (nt) sequence elements involved in the E1a-responsiveness of the E2aE transcription unit: element I, localized within a segment (nt -13 to +62) surrounding the major E2aE cap site (nt + 1); element II (between nt -71 and -29), and element III (between nt -146 and -86). Each element is unable by itself to confer E1a responsiveness. Only combinations of sequences including elements I and II (spanning nt -71 and +62) or II and III (spanning nt -146 and -29) ensure maximal inducibility, for which element II appears of central importance.

Different estrogen receptor structural domains are required for estrogen- and tamoxifen-dependent anti-proliferative activity in human mammary epithelial cells expressing an exogenous estrogen receptor.

Estrogen (E) inhibits the growth of both non-tumorigenic, immortal human mammary epithelial cells (HMEC) and breast cancer cells which stably express exogenous estrogen receptors (ER). The anti-estrogenic compounds 4-hydroxy-tamoxifen (HT) and ICI 164384 (ICI) have different effects on the growth of the ER-transfectants. HT is a potent growth inhibitor, while ICI has no effect by itself but is able to block the anti-proliferative effects of E and HT. In order to elucidate the mechanism by which E or HT-bound ER inhibit cell growth, we have evaluated the effects of these compounds on the growth of HMEC stably expressing ER with mutations or deletions in the N-terminal A/B domain, the DNA-binding domain (DBD), and the C-terminal ligand-binding domain. These studies revealed that E and HT require different structural domains of the ER for their anti-proliferative activities. The N-terminal A/B domain is required for HT-, but not E-dependent growth inhibition. The DNA-binding domain of the ER is not essential for HT-mediated anti-proliferative effects, but is important for E-dependent activity. The effect of ER mutations on the ligand-inducible expression of the endogenous progesterone receptor (PR) and pS2 genes was also evaluated. Neither gene was induced in the cells containing the ER mutated in the DBD, even though cell growth was inhibited. These results suggest that E and HT use different pathways to elicit their anti-proliferative effects and that this occurs via modulation of genes that are controlled by mechanisms different from those important for activation of the PR and pS2 genes.

Functional diversity of gro gene expression in human fibroblasts and mammary epithelial cells.

Previous studies of gro and related genes that are overexpressed in transformed fibroblasts suggest that gro may encode a specific growth regulator. However, DNA and protein sequence comparisons reveal relatedness to platelet factor 4 and other proteins involved in the inflammatory response. In this paper, both growth-related and cytokine-induced responses in gro gene expression are described. Human foreskin fibroblasts are shown to express approximately 10-fold elevated gro, myc, and fos mRNAs in response to serum and to phorbol 12-myristate 13-acetate stimulation, with early response kinetics indicative of growth regulation. In response to interleukin 1, however, in growing cells gro mRNA is elevated at least 100-fold but myc remains constant and fos is not expressed, suggesting a second regulatory pathway. In normal cultured mammary epithelial cells, gro is constitutively expressed, and elevated mRNA levels are induced by phorbol 12-myristate 13-acetate, but not by interleukin 1. However, most carcinoma cell lines examined do not express gro mRNA, suggesting a third function of gro as a negative growth regulator in epithelial cells.

EIa-mediated stimulation of the adenovirus EIII promoter involves an enhancer element within the nearby EIIa promoter.

The transcriptional induction of adenovirus early genes by the viral immediate early gene EIa constitutes an attractive model system for the study of control mechanisms involved in eucaryotic promoter function. The EIa-mediated activation of the divergently transcribed EIIa early (EIIaE) and EIII promoters was investigated in experiments in which recombinant plasmids containing the entire EIIa-EIII control region were cotransfected with a plasmid expressing the EIa 13S mRNA. First, both promoters were activated by low levels of EIa, but the extent of EIII induction decreased with increasing EIa concentrations, whereas EIIaE stimulation remained unchanged. Second, transcriptional analysis of deletion mutants revealed that an element of the EIIaE promoter contributed to maximal EIa responsiveness of the nearby EIII promoter. This element, located between positions -82 and -71 with respect to the EIIaE major cap site, corresponded to the central portion of an EIa-dependent enhancer, originally mapped between about -110 and -50 (P. Jalinot and C. Kédinger, Nucleic Acids Res. 14:2651-2669, 1986). The implication of these observations in the coordinate expression from the EIIaE and EIII promoters during lytic infection is discussed.

Human papilloma virus DNAs immortalize normal human mammary epithelial cells and reduce their growth factor requirements.

Human papilloma virus (HPV) types 16 and 18 are most commonly associated with cervical carcinoma in patients and induce immortalization of human keratinocytes in culture. HPV has not been associated with breast cancer. This report describes the immortalization of normal human mammary epithelial cells (76N) by plasmid pHPV18 or pHPV16, each containing the linearized viral genome. Transfectants were grown continuously for more than 60 passages, whereas 76N cells senesce after 18-20 passages. The transfectants also differ from 76N cells in cloning in a completely defined medium called D2 and growing in a minimally supplemented defined medium (D3) containing epidermal growth factor. All transfectants tested contain integrated HPV DNA, express HPV RNA, and produce HPV E7 protein. HPV transfectants do not form tumors in a nude mouse assay. It is concluded that products of the HPV genome induce immortalization of human breast epithelial cells and reduce their growth factor requirements. This result raises the possibility that HPV might be involved in breast cancer. Furthermore, other tissue-specific primary epithelial cells that are presently difficult to grow and investigate may also be immortalized by HPV.

The adenovirus-2 early EIIa transcription unit possesses two overlapping promoters with different sequence requirements for EIa-dependent stimulation.

The EIa-inducible, EIIa transcription unit of adenovirus-2 is transcribed early in infection from two start sites (+1 or EIIaE1 and -26 or EIIaE2), neither of which is preceded by canonical TATA box elements. Analysis of promoter deletion and linker scanning mutations for in vivo transcriptional activity after transfection into HeLa cells has indicated the existence of two overlapping promoters in the EIIaE gene. Two regions, each approximately 30 nucleotides upstream from start sites EIIaE1 and EIIaE2, function as TATA box substitutes. A sequence centered at position -42 (with respect to the major start site at position +1) is essential for transcription from both sites, while an element further upstream, localized between nucleotides -91 and -62, is also required for efficient EIIaE transcription, with the 3' border being dispensable for EIIaE2 transcription. Analysis of the entire series of EIIaE mutants, co-transfected with an EIa-containing plasmid, revealed that no unique sequence elements in the EIIaE1 promoter region between -97 and +1 were responsible for the stimulation of EIIaE1 transcription by EIa. In contrast, the EIa-mediated augmentation of EIIaE2 template activity was mainly dependent upon a sequence, the 5'-TTAAATTT-3' putative TATA box substitute, located around position -59.

Specific cellular proteins bind to critical promoter sequences of the adenovirus early EIIa promoter.

As an approach to the identification of essential factors required for specific expression of the adenovirus type 2 EIIaE early (EIIaE) promoter, an in vitro system was established. Under appropriate conditions, using crude extracts of non-infected HeLa cells, efficient and accurate EIIaE expression has been reproduced. As in vivo, this transcription was strongly dependent upon the integrity of two non-consensus TATA-like elements, T1 and T2, corresponding to the major (EIIaE1) and minor (EIIaE2) start sites, respectively, as well as upon intact upstream elements (A, between -40 and -50 and B, between -70 and -90) common to both overlapping promoters. The implication of specific DNA-binding proteins in the transcriptional effects mediated by these elements was demonstrated by DNAse I footprinting analyses. Both crude nuclear extracts and partially purified fractions confer specific protection against DNAse I digestion to the T1 and B promoter elements defined above, and to a far upstream region (element C, between -110 and -150), which has previously been identified as a weaker promoter element by in vivo transcriptional studies. Separation of the T1 recognition factor from those which bind to the upstream elements B and C by chromatographic fractionation of the extracts has also been achieved.

Protein fold analysis of the B30.2-like domain.

The B30.2-like domain occurs in some members of a diverse and growing family of proteins containing zinc-binding B-box motifs, whose functions include regulation of cell growth and differentiation. The B30.2-like domain is also found in proteins without the zinc-binding motifs, such as butyrophilin (a transmembrane glycoprotein) and stonustoxin (a secreted cytolytic toxin). Currently, the function for the B30.2-like domain is not clear and the structure of a protein containing this domain has not been solved. The secondary structure prediction methods indicate that the B30.2-like domain consists of fifteen or fewer beta-strands. Fold recognition methods identified different structural topologies for the B30.2-like domains. Secondary structure prediction, deletion and lack of local sequence identity at the C-terminal region for certain members of the family, and packing of known core structures suggest that a structure containing two beta domains is the most probable of these folds. The most C-terminal sequence motif predicted to be a beta-strand in all B30.2-like domains is a potential subdomain boundary based on the sequence-structure alignments. Models of the B30.2-like domains were built based on immunoglobulin-like folds identified by the fold recognition methods to evaluate the possibility of the B30.2 domain adopting known folds and infer putative functional sites. The SPRY domain has been identified as a subdomain within the B30.2-like domain. If the B30.2-like domain is a subclass of the SPRY domain family, then this analysis would suggest that the SPRY domains are members of the immunoglobulin superfamily.

Estrogen modulation of apolipoprotein(a) expression. Identification of a regulatory element.

Elevated plasma levels of the lipoprotein particle Lp(a) are a major risk factor for cardiovascular disease. Lp(a) plasma levels are determined by the level of expression of its characteristic protein component, apo(a). Apo(a) expression is modulated by several hormones, of which estrogens are the best known. The chromosomal region responsible for estrogen response was identified within an apo(a) enhancer located at approximately 26 kilobases from the apo(a) promoter. Although the estrogen-responsive unit contains a potential estrogen response element, binding of estrogen receptor-alpha to DNA was not necessary. The receptor, activated by bound estradiol, interacts through its transactivation domains with a transcription factor necessary for the function of the enhancer, preventing its binding to DNA.

Keratins as markers that distinguish normal and tumor-derived mammary epithelial cells.

Keratin 5 (K5) mRNA and protein are shown to be expressed in normal mammary epithelial cells in culture and are absent from tumor-derived cell lines. To extend these findings, the full complements of keratins in normal, immortalized, and tumor cells were compared. It is shown here that normal cells produce keratins K5, K6, K7, K14, and K17, whereas tumor cells produce mainly keratins K8, K18, and K19. In immortalized cells, which are preneoplastic or partially transformed, the levels of K5 mRNA and protein are lower than in normal cells, whereas the amount of K18 is increased. Thus, K5 is an important marker in the tumorigenic process, distinguishing normal from tumor cells, and decreased K5 expression correlates with tumorigenic progression.

Sequence-specific trans-activation of the adenovirus EIIa early promoter by the viral EIV transcription unit.

The contribution of adenovirus early genes, other than that of the well-documented EIa immediate early gene, to the transcriptional regulation of the viral EIIa early transcription unit was examined. HeLa cells were transfected with EIIa-containing plasmids and co-transfected with distinct plasmids bearing one of the viral regions EIa, EIII or EIV. Co-transfection with the EIV-recombinants, but not the EIII constructs, stimulated specific transcription from the major EIIaE start site (EIIaE1) by 5- to 15-fold, as concluded from quantitative S1 nuclease analysis of cytoplasmic RNA and in vitro nuclear 'run-on' transcription assays. The extent of the EIV-induced stimulation was similar to that achieved by EIa under identical conditions. However, in contrast to our observations for EIa-mediated stimulation, where no unique EIIaE1 promoter elements were implicated, maximal induction by EIV requires sequences between positions -48 and -19 (with respect to the EIIaE1 start site).

A newly established metastatic breast tumor cell line with integrated amplified copies of ERBB2 and double minute chromosomes.

A continuous line of human mammary tumor cells, called 21MT, has been established in culture from a pleural effusion of a 36-year-old woman with metastatic breast cancer. The cells are epithelial as shown by morphology and expression of keratins and are mammary tumor cells as shown by expression of the HMFG-2 antigenic determinant. The cells grow well both in DFCI-1, a partially defined medium containing pituitary extract and 1% fetal bovine serum, and in alpha-minimum essential medium (alpha-MEM) supplemented with 10% serum, epidermal growth factor (EGF), insulin, and hydrocortisone. Karyotypic analysis of cells at early passage has shown the presence of rearranged (marker) chromosomes as well as aneuploidy with a net DNA content in the tetraploid range, confirmed by DNA cytofluorography, as well as double minute chromosomes in about 5% of the cells. Southern blots have revealed a 40-fold amplification of the ERBB2 gene and a 50-fold overexpression of its mRNA. The amplification of ERBB2 DNA was localized by in situ hybridization to one of the marker chromosomes but not to the double minutes. It is inferred, therefore, that at least two genes have been amplified in these cells.

Proteinase inhibitor 9, an inhibitor of granzyme B-mediated apoptosis, is a primary estrogen-inducible gene in human liver cells.

Although liver is an estrogen target tissue, the number of hepatic genes known to be directly induced by estrogen is very small. We identified proteinase inhibitor 9, or PI-9, as being rapidly and strongly induced by estrogen in an estrogen receptor-positive human liver cell line (HepG2-ER7). Since PI-9 mRNA was also induced by estrogen in a human liver biopsy sample, PI-9 is a genuine estrogen-regulated human gene. PI-9 is a potent inhibitor of granzyme B and of granzyme B-mediated apoptosis. Estrogens induced PI-9 mRNA within 2 h, PI-9 mRNA levels reached a plateau of 30-40-fold induction in 4 h, and induction was not blocked by cycloheximide, indicating that induction of PI-9 mRNA is a primary response. The antiestrogen trans-hydroxytamoxifen was a partial agonist for PI-9 mRNA induction, whereas the antiestrogen ICI 182, 780 was a pure antagonist. Western blot analysis showed that estrogen strongly increases PI-9 protein levels. Inhibition of transcription with actinomycin D resulted in identical rates of PI-9 mRNA decay in the presence and absence of estrogen. We isolated genomic clones containing the PI-9 promoter region, identified a putative transcription start site, and carried out transient transfections of PI-9-luciferase reporter gene constructs. The estrogen, moxestrol, elicited a robust induction from the PI-9-luciferase reporter. Mutational inactivation of three potential imperfect estrogen response elements in the PI-9 5'-flanking region had no effect on moxestrol estrogen receptor induction.