High-sensitivity array analysis of gene expression for the early detection of disseminated breast tumor cells in peripheral blood

Early detection is an effective means of reducing cancer mortality. Here, we describe a highly sensitive high-throughput screen that can identify panels of markers for the early detection of solid tumor cells disseminated in peripheral blood. The method is a two-step combination of differential display and high-sensitivity cDNA arrays. In a primary screen, differential display identified 170 candidate marker genes differentially expressed between breast tumor cells and normal breast epithelial cells. In a secondary screen, high-sensitivity arrays assessed expression levels of these genes in 48 blood samples, 22 from healthy volunteers and 26 from breast cancer patients. Cluster analysis identified a group of 12 genes that were elevated in the blood of cancer patients. Permutation analysis of individual genes defined five core genes (P ≤ 0.05, permax test). As a group, the 12 genes generally distinguished accurately between healthy volunteers and patients with breast cancer. Mean expression levels of the 12 genes were elevated in 77% (10 of 13) untreated invasive cancer patients, whereas cluster analysis correctly classified volunteers and patients (P = 0.0022, Fisher's exact test). Quantitative real-time PCR confirmed array results and indicated that the sensitivity of the assay (1:2 × 108 transcripts) was sufficient to detect disseminated solid tumor cells in blood. Expression-based blood assays developed with the screening approach described here have the potential to detect and classify solid tumor cells originating from virtually any primary site in the body.

Analysis of genetic instability during mammary tumor progression using a novel selection-based assay for in vivo mutations in a bacteriophage lambda transgene target.

Genetic instability is thought to be responsible for the numerous genotypic changes that occur during neoplastic transformation and metastatic progression. To explore the role of genetic instability at the level of point mutations during mammary tumor development and malignant progression, we combined transgenic mouse models of mutagenesis detection and oncogenesis. Bitransgenic mice were generated that carried both a bacteriophage lambda transgene to assay mutagenesis and a polyomavirus middle T oncogene, mammary gland-targeted expression of which led to metastatic mammary adenocarcinomas. We developed a novel assay for the detection of mutations in the lambda transgene that selects for phage containing forward mutations only in the lambda cII gene, using an hfl- bacterial host. In addition to the relative ease of direct selection, the sensitivity of this assay for both spontaneous and chemically induced mutations was comparable to the widely used mutational target gene, lambda lacI, making the cII assay an attractive alternative for mutant phage recovery for any lambda-based mouse mutagenesis assay system. The frequencies of lambda cII- mutants were not significantly different in normal mammary epithelium, primary mammary adenocarcinomas, and pulmonary metastases. The cII mutational spectra in these tissues consisted mostly of G/C-->A/T transitions, a large fraction of which occurred at CpG dinucleotides. These data suggest that, in this middle T oncogene model of mammary tumor progression, a significant increase in mutagenesis is not required for tumor development or for metastatic progression.

Genetic analysis using genomic representations

Analysis of the genetic changes in human tumors is often problematical because of the presence of normal stroma and the limited availability of pure tumor DNA. However, large amounts of highly reproducible “representations” of tumor and normal genomes can be made by PCR from nanogram amounts of restriction endonuclease cleaved DNA that has been ligated to oligonucleotide adaptors. We show here that representations are useful for many types of genetic analyses, including measuring relative gene copy number, loss of heterozygosity, and comparative genomic hybridization. Representations may be prepared even from sorted nuclei from fixed and archived tumor biopsies.

HLA-G expression in melanoma: A way for tumor cells to escape from immunosurveillance

Considering the well established role of nonclassical HLA-G class I molecules in inhibiting natural killer (NK) cell function, the consequence of abnormal HLA-G expression in malignant cells should be the escape of tumors from immunosurveillance. To examine this hypothesis, we analyzed HLA-G expression and NK sensitivity in human malignant melanoma cells. Our analysis of three melanoma cell lines and ex vivo biopsy demonstrated that (i) IGR and M74 human melanoma cell lines exhibit a high level of HLA-G transcription with differential HLA-G isoform transcription and protein expression patterns, (ii) a higher level of HLA-G transcription ex vivo is detected in a skin melanoma metastasis biopsy compared with a healthy skin fragment from the same individual, and (iii) HLA-G protein isoforms other than membrane-bound HLA-G1 protect IGR from NK lysis. It thus appears of critical importance to consider the specific role of HLA-G expression in tumors in the design of future cancer immunotherapies.

Inhibition of DNA methyltransferase stimulates the expression of signal transducer and activator of transcription 1, 2, and 3 genes in colon tumor cells

Inhibitors of DNA methyltransferase, typified by 5-aza-2′-deoxycytidine (5-Aza-CdR), induce the expression of genes transcriptionally down-regulated by de novo methylation in tumor cells. We utilized gene expression microarrays to examine the effects of 5-Aza-CdR treatment in HT29 colon adenocarcinoma cells. This analysis revealed the induction of a set of genes that implicated IFN signaling in the HT29 cellular response to 5-Aza-CdR. Subsequent investigations revealed that the induction of this gene set correlates with the induction of signal transducer and activator of transcription (STAT) 1, 2, and 3 genes and their activation by endogenous IFN-α. These observations implicate the induction of the IFN-response pathway as a major cellular response to 5-Aza-CdR and suggests that the expression of STATs 1, 2, and 3 can be regulated by DNA methylation. Consistent with STAT’s limiting cell responsiveness to IFN, we found that 5-Aza-CdR treatment sensitized HT29 cells to growth inhibition by exogenous IFN-α2a, indicating that 5-Aza-CdR should be investigated as a potentiator of IFN responsiveness in certain IFN-resistant tumors.

A subset of skin tumor modifier loci determines survival time of tumor-bearing mice

Studies of mouse models of human cancer have established the existence of multiple tumor modifiers that influence parameters of cancer susceptibility such as tumor multiplicity, tumor size, or the probability of malignant progression. We have carried out an analysis of skin tumor susceptibility in interspecific Mus musculus/Mus spretus hybrid mice and have identified another seven loci showing either significant (six loci) or suggestive (one locus) linkage to tumor susceptibility or resistance. A specific search was carried out for skin tumor modifier loci associated with time of survival after development of a malignant tumor. A combination of resistance alleles at three markers [D6Mit15 (Skts12), D7Mit12 (Skts2), and D17Mit7 (Skts10)], all of which are close to or the same as loci associated with carcinoma incidence and/or papilloma multiplicity, is significantly associated with increased survival of mice with carcinomas, whereas the reverse combination of susceptibility alleles is significantly linked to early mortality caused by rapid carcinoma growth (χ2 = 25.22; P = 5.1 × 10−8). These data indicate that host genetic factors may be used to predict carcinoma growth rate and/or survival of individual backcross mice exposed to the same carcinogenic stimulus and suggest that mouse models may provide an approach to the identification of genetic modifiers of cancer survival in humans.

Genomic and cDNA sequence analysis of the cell matrix adhesion regulator gene

The cell matrix adhesion regulator (CMAR) gene has been suggested to be a signal transduction molecule influencing cell adhesion to collagen and, through this, possibly involved in tumor suppression. The originally reported CMAR cDNA was 464 bp long with a tyrosine phosphorylation site at the extreme 3′ end, which mutagenesis studies had shown to be central to the function of this gene. Since the discovery of a 4-bp insertion polymorphism within the originally reported coding region, further sequence information has been obtained. The cDNA has been extended 5′ by ≈2 kb revealing a 559-bp region showing strong homology to the proposed 5′ untranslated sequence of a murine protein kinase receptor family member, variant in kinase (vik). CMAR genomic sequencing has shown the presence of an intron, the intron/exon boundary lying within this region of homology. An RNA transcript for CMAR of ≈2.5 kb has also been identified. The data suggest complex mechanisms for control of expression of two closely associated genes, CMAR and the vik- associated sequence.

Analysis of genomic alterations in benign, atypical, and anaplastic meningiomas: Toward a genetic model of meningioma progression

Nineteen benign [World Health Organization (WHO) grade I; MI], 21 atypical (WHO grade II; MII), and 19 anaplastic (WHO grade III; MIII) sporadic meningiomas were screened for chromosomal imbalances by comparative genomic hybridization (CGH). These data were supplemented by molecular genetic analyses of selected chromosomal regions and genes. With increasing malignancy grade, a marked accumulation of genomic aberrations was observed; i.e., the numbers (mean ± SEM) of total alterations detected per tumor were 2.9 ± 0.7 for MI, 9.2 ± 1.2 for MII, and 13.3 ± 1.9 for MIII. The most frequent alteration detected in MI was loss on 22q (58%). In MII, aberrations most commonly identified were losses on 1p (76%), 22q (71%), 14q (43%), 18q (43%), 10 (38%), and 6q (33%), as well as gains on 20q (48%), 12q (43%), 15q (43%), 1q (33%), 9q (33%), and 17q (33%). In MIII, most of these alterations were found at similar frequencies. However, an increase in losses on 6q (53%), 10 (68%), and 14q (63%) was observed. In addition, 32% of MIII demonstrated loss on 9p. Homozygous deletions in the CDKN2A gene at 9p21 were found in 4 of 16 MIII (25%). Highly amplified DNA sequences were mapped to 12q13–q15 by CGH in 1 MII. Southern blot analysis of this tumor revealed amplification of CDK4 and MDM2. By CGH, DNA sequences from 17q were found to be amplified in 1 MII and 8 MIII, involving 17q23 in all cases. Despite the high frequency of chromosomal aberrations in the MII and MIII investigated, none of these tumors showed mutations in exons 5–8 of the TP53 gene. On the basis of the most common aberrations identified in the various malignancy grades, a model for the genomic alterations associated with meningioma progression is proposed.

Combined effect of tumor necrosis factor-related apoptosis-inducing ligand and ionizing radiation in breast cancer therapy

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a potent endogenous activator of the cell death pathway and functions by activating the cell surface death receptors 4 and 5 (DR4 and DR5). TRAIL is nontoxic in vivo and preferentially kills neoplastically transformed cells over normal cells by an undefined mechanism. Radiotherapy is a common treatment for breast cancer as well as many other cancers. Here we demonstrate that ionizing radiation can sensitize breast carcinoma cells to TRAIL-induced apoptosis. This synergistic effect is p53-dependent and may be the result of radiation-induced up-regulation of the TRAIL-receptor DR5. Importantly, TRAIL and ionizing radiation have a synergistic effect in the regression of established breast cancer xenografts. Changes in tumor cellularity and extracellular space were monitored in vivo by diffusion-weighted magnetic resonance imaging (diffusion MRI), a noninvasive technique to produce quantitative images of the apparent mobility of water within a tissue. Increased water mobility was observed in combined TRAIL- and radiation-treated tumors but not in tumors treated with TRAIL or radiation alone. Histological analysis confirmed the loss of cellularity and increased numbers of apoptotic cells in TRAIL- and radiation-treated tumors. Taken together, our results provide support for combining radiation with TRAIL to improve tumor eradication and suggest that efficacy of apoptosis-inducing cancer therapies may be monitored noninvasively, using diffusion MRI.

A male germ cell tumor-susceptibility-determining locus, pgct1, identified on murine chromosome 13

Inbred 129 strain mice are predisposed to developing male germ cell tumors (GCTs) of the testes. The inherent genetic defects that underlie male GCT susceptibility in the 129 mouse strain are unknown. GCT incidence is increased in 129 strain males that lack functional p53 protein, and we have used this finding to facilitate the generation of panels of GCT-bearing intercross and backcross mice for genetic mapping analysis. A 129 strain locus, designated pgct1, that segregates with the male GCT phenotype has been identified on chromosome 13 near D13Mit188. This region of murine chromosome 13 may be syntenic to a portion of human chromosome 5q that is implicated in male GCT susceptibility in humans.

Differential gene expression in p53-mediated apoptosis-resistant vs. apoptosis-sensitive tumor cell lines

Induction of wild-type p53 in the ECV-304 bladder carcinoma cell line by infection with a p53 recombinant adenovirus (Ad5CMV-p53) resulted in extensive apoptosis and eventual death of nearly all of the cells. As a strategy to determine the molecular events important to p53-mediated apoptosis in these transformed cells, ECV-304 cells were selected for resistance to p53 by repeated infections with Ad5CMV-p53. We compared the expression of 5,730 genes in p53-resistant (DECV) and p53-sensitive ECV-304 cells by reverse transcription–PCR, Northern blotting, and DNA microarray analysis. The expression of 480 genes differed by 2-fold or more between the two p53-infected cell lines. A number of potential targets for p53 were identified that play roles in cell cycle regulation, DNA repair, redox control, cell adhesion, apoptosis, and differentiation. Proline oxidase, a mitochondrial enzyme involved in the proline/pyrroline-5-carboxylate redox cycle, was up-regulated by p53 in ECV but not in DECV cells. Pyrroline-5-carboxylate (P5C), a proline-derived metabolite generated by proline oxidase, inhibited the proliferation and survival of ECV-304 and DECV cells and induced apoptosis in both cell lines. A recombinant proline oxidase protein tagged with a green fluorescent protein at the amino terminus localized to mitochondria and induced apoptosis in p53-null H1299 non-small cell lung carcinoma cells. The results directly implicate proline oxidase and the proline/P5C pathway in p53-induced growth suppression and apoptosis.

Endothelial cell death, angiogenesis, and microvascular function after castration in an androgen-dependent tumor: Role of vascular endothelial growth factor

The sequence of events that leads to tumor vessel regression and the functional characteristics of these vessels during hormone–ablation therapy are not known. This is because of the lack of an appropriate animal model and monitoring technology. By using in vivo microscopy and in situ molecular analysis of the androgen-dependent Shionogi carcinoma grown in severe combined immunodeficient mice, we show that castration of these mice leads to tumor regression and a concomitant decrease in vascular endothelial growth factor (VEGF) expression. Androgen withdrawal is known to induce apoptosis in Shionogi tumor cells. Surprisingly, tumor endothelial cells begin to undergo apoptosis before neoplastic cells, and rarefaction of tumor vessels precedes the decrease in tumor size. The regressing vessels begin to exhibit normal phenotype, i.e., lower diameter, tortuosity, vascular permeability, and leukocyte adhesion. Two weeks after castration, a second wave of angiogenesis and tumor growth begins with a concomitant increase in VEGF expression. Because human tumors often relapse following hormone–ablation therapy, our data suggest that these patients may benefit from combined anti-VEGF therapy.

Cytologically normal cells from neoplastic cervical samples display extensive structural abnormalities on IR spectroscopy: Implications for tumor biology

Fourier-transform IR (FT-IR) spectra of pelleted exfoliated cervical cells from patients with cervical cancer or dysplasia differ from those from normal women. To study the origin of these spectral changes, we obtained the FT-IR spectra of individual cervical cells from normal, dysplastic, and malignant cervical samples. Ninety five percent of normal superficial and intermediate cells displayed two distinct spectral patterns designated A and B, and 5% displayed an intermediate pattern, suggesting extensive structural heterogeneity among these cells. Parabasal and endocervical cells showed pattern B spectra. The spectra of malignant, dysplastic, and other abnormal cells also were characterized. Analysis of FT-IR spectra of over 2,000 individual cells from 10 normal females, 7 females with dysplasia, and 5 females with squamous cell carcinoma revealed that the spectra of normal-appearing intermediate and superficial cells of the cervix from women with either dysplasia or cancer differed from those of normal women. Chemometric and classical spectroscopic analysis showed a continuum of changes paralleling the transition from normalcy to malignancy. These findings suggest that (i) the structural changes underlying the spectroscopic changes are involved in or are a product of cervical carcinogenesis and (ii) the neoplastic process may be more extensive than currently recognized with morphological criteria. This approach may be useful for the structural study of neoplasia and also may be of help in the diagnosis or classification of cervical disorders.

Gene transfer of Fas ligand induces tumor regression in vivo

The Fas–Fas ligand (FasL) system plays an important role in the induction of lymphoid apoptosis and has been implicated in the suppression of immune responses. Herein, we report that gene transfer of FasL inhibits tumor cell growth in vivo. Although such inhibition is expected in Fas+ tumor cell lines, marked regression was unexpectedly observed after FasL gene transfer into the CT26 colon carcinoma that does not express Fas. Infection by an adenoviral vector encoding FasL rapidly eliminated tumor masses in the Fas+ Renca tumor by inducing cell death, whereas the elimination of Fas− CT26 cells was mediated by inflammatory cells. Analysis of human malignancies revealed Fas, but not FasL, expression in a majority of tumors and susceptibility to FasL in most Fas+ cell lines. These findings suggest that gene transfer of FasL generates apoptotic responses and induces potent inflammatory reactions that can be used to induce the regression of malignancies.

Polyclonal structure of intestinal adenomas in ApcMin/+ mice with concomitant loss of Apc+ from all tumor lineages

When tumors form in intestinal epithelia, it is important to know whether they involve single initiated somatic clones. Advanced carcinomas in humans and mice are known to be monoclonal. However, earlier stages of tumorigenesis may instead involve an interaction between cells that belong to separate somatic clones within the epithelium. The clonality of early tumors has been investigated in mice with an inherited predisposition to intestinal tumors. Analysis of Min (multiple intestinal neoplasia) mice chimeric for a ubiquitously expressed cell lineage marker revealed that normal intestinal crypts are monoclonal, but intestinal adenomas frequently have a polyclonal structure, presenting even when very small as single, focal adenomas composed of at least two somatic lineages. Furthermore, within these polyclonal adenomas, all tumor lineages frequently lose the wild-type Apc allele. These observations can be interpreted by several models for clonal interaction within the epithelium, ranging from passive fusion within regions of high neoplastic potential to a requirement for active clonal cooperation.