Impact of centralization on aCGH-based genomic profiles for precision medicine in oncology.

BACKGROUND: Comparative genomic hybridization (CGH) arrays are increasingly used in personalized medicine programs to identify gene copy number aberrations (CNAs) that may be used to guide clinical decisions made during molecular tumor boards. However, analytical processes such as the centralization step may profoundly affect CGH array results and therefore may adversely affect outcomes in the precision medicine context. PATIENTS AND METHODS: The effect of three different centralization methods: median, maximum peak, alternative peak, were evaluated on three datasets: (i) the NCI60 cell lines panel, (ii) the Cancer Cell Line Encyclopedia (CCLE) panel, and (iii) the patients enrolled in prospective molecular screening trials (SAFIR-01 n = 283, MOSCATO-01 n = 309), and compared with karyotyping, drug sensitivity, and patient-drug matching, respectively. RESULTS: Using the NCI60 cell lines panel, the profiles generated by the alternative peak method were significantly closer to the cell karyotypes than those generated by the other centralization strategies (P < 0.05). Using the CCLE dataset, selected genes (ERBB2, EGFR) were better or equally correlated to the IC50 of their companion drug (lapatinib, erlotinib), when applying the alternative centralization. Finally, focusing on 24 actionable genes, we observed as many as 7.1% (SAFIR-01) and 6.8% (MOSCATO-01) of patients originally not oriented to a specific treatment, but who could have been proposed a treatment based on the alternative peak centralization method. CONCLUSION: The centralization method substantially affects the call detection of CGH profiles and may thus impact precision medicine approaches. Among the three methods described, the alternative peak method addresses limitations associated with existing approaches.

Screening patients for heterozygous p53 mutations using a functional assay in yeast.

Inherited mutations of the p53 gene significantly increase the risk of developing diverse malignancies, and germline p53 mutations can be detected by assaying the transcriptional activity of the p53 protein in mammalian cells. Here we describe a method starting with lymphocytes that allows detection of germline p53 mutations by 'functional' analysis of p53 protein expressed in Saccharomyces cerevisiae. The p53 PCR products are directly cloned into yeast expression vectors in vivo and subsequently tested for transcriptional activity in a simple growth assay. This technique, functional analysis of separated alleles in yeast (FASAY), requires only a few steps, can be automated readily and should permit screening for germline or somatic heterozygous mutations in any gene whose function can be monitored in yeast.

Functional analysis of human MLH1 mutations in Saccharomyces cerevisiae.

Hereditary non-polyposis colorectal cancer (HNPCC; OMIM 120435-6) is a cancer-susceptibility syndrome linked to inherited defects in human mismatch repair (MMR) genes. Germline missense human MLH1 (hMLH1) mutations are frequently detected in HNPCC (ref. 3), making functional characterization of mutations in hMLH1 critical to the development of genetic testing for HNPCC. Here, we describe a new method for detecting mutations in hMLH1 using a dominant mutator effect of hMLH1 cDNA expressed in Saccharomyces cerevisiae. The majority of hMLH1 missense mutations identified in HNPCC patients abolish the dominant mutator effect. Furthermore, PCR amplification of hMLH1 cDNA from mRNA from a HNPCC patient, followed by in vivo recombination into a gap expression vector, allowed detection of a heterozygous loss-of-function missense mutation in hMLH1 using this method. This functional assay offers a simple method for detecting and evaluating pathogenic mutations in hMLH1.

Widespread aneuploidy revealed by DNA microarray expression profiling.

Expression profiling using DNA microarrays holds great promise for a variety of research applications, including the systematic characterization of genes discovered by sequencing projects. To demonstrate the general usefulness of this approach, we recently obtained expression profiles for nearly 300 Saccharomyces cerevisiae deletion mutants. Approximately 8% of the mutants profiled exhibited chromosome-wide expression biases, leading to spurious correlations among profiles. Competitive hybridization of genomic DNA from the mutant strains and their isogenic parental wild-type strains showed they were aneuploid for whole chromosomes or chromosomal segments. Expression profile data published by several other laboratories also suggest the use of aneuploid strains. In five separate cases, the extra chromosome harboured a close homologue of the deleted gene; in two cases, a clear growth advantage for cells acquiring the extra chromosome was demonstrated. Our results have implications for interpreting whole-genome expression data, particularly from cells known to suffer genomic instability, such as malignant or immortalized cells.

Drug target validation and identification of secondary drug target effects using DNA microarrays.

We describe here a method for drug target validation and identification of secondary drug target effects based on genome-wide gene expression patterns. The method is demonstrated by several experiments, including treatment of yeast mutant strains defective in calcineurin, immunophilins or other genes with the immunosuppressants cyclosporin A or FK506. Presence or absence of the characteristic drug 'signature' pattern of altered gene expression in drug-treated cells with a mutation in the gene encoding a putative target established whether that target was required to generate the drug signature. Drug dependent effects were seen in 'targetless' cells, showing that FK506 affects additional pathways independent of calcineurin and the immunophilins. The described method permits the direct confirmation of drug targets and recognition of drug-dependent changes in gene expression that are modulated through pathways distinct from the drug's intended target. Such a method may prove useful in improving the efficiency of drug development programs.

Integrating genetic approaches into the discovery of anticancer drugs.

The discovery of anticancer drugs is now driven by the numerous molecular alterations identified in tumor cells over the past decade. To exploit these alterations, it is necessary to understand how they define a molecular context that allows increased sensitivity to particular compounds. Traditional genetic approaches together with the new wealth of genomic information for both human and model organisms open up strategies by which drugs can be profiled for their ability to selectively kill cells in a molecular context that matches those found in tumors. Similarly, it may be possible to identify and validate new targets for drugs that would selectively kill tumor cells with a particular molecular context. This article outlines some of the ways that yeast genetics can be used to streamline anticancer drug discovery.

Genetic selection of peptide inhibitors of biological pathways.

Genetic selections were used to find peptides that inhibit biological pathways in budding yeast. The peptides were presented inside cells as peptamers, surface loops on a highly expressed and biologically inert carrier protein, a catalytically inactive derivative of staphylococcal nuclease. Peptamers that inhibited the pheromone signaling pathway, transcriptional silencing, and the spindle checkpoint were isolated. Putative targets for the inhibitors were identified by a combination of two-hybrid analysis and genetic dissection of the target pathways. This analysis identified Ydr517w as a component of the spindle checkpoint and reinforced earlier indications that Ste50 has both positive and negative roles in pheromone signaling. Analysis of transcript arrays showed that the peptamers were highly specific in their effects, which suggests that they may be useful reagents in organisms that lack sophisticated genetics as well as for identifying components of existing biological pathways that are potential targets for drug discovery.

Signaling and circuitry of multiple MAPK pathways revealed by a matrix of global gene expression profiles.

Genome-wide transcript profiling was used to monitor signal transduction during yeast pheromone response. Genetic manipulations allowed analysis of changes in gene expression underlying pheromone signaling, cell cycle control, and polarized morphogenesis. A two-dimensional hierarchical clustered matrix, covering 383 of the most highly regulated genes, was constructed from 46 diverse experimental conditions. Diagnostic subsets of coexpressed genes reflected signaling activity, cross talk, and overlap of multiple mitogen-activated protein kinase (MAPK) pathways. Analysis of the profiles specified by two different MAPKs-Fus3p and Kss1p-revealed functional overlap of the filamentous growth and mating responses. Global transcript analysis reflects biological responses associated with the activation and perturbation of signal transduction pathways.

An information-intensive approach to the molecular pharmacology of cancer.

Since 1990, the National Cancer Institute (NCI) has screened more than 60,000 compounds against a panel of 60 human cancer cell lines. The 50-percent growth-inhibitory concentration (GI50) for any single cell line is simply an index of cytotoxicity or cytostasis, but the patterns of 60 such GI50 values encode unexpectedly rich, detailed information on mechanisms of drug action and drug resistance. Each compound's pattern is like a fingerprint, essentially unique among the many billions of distinguishable possibilities. These activity patterns are being used in conjunction with molecular structural features of the tested agents to explore the NCI's database of more than 460,000 compounds, and they are providing insight into potential target molecules and modulators of activity in the 60 cell lines. For example, the information is being used to search for candidate anticancer drugs that are not dependent on intact p53 suppressor gene function for their activity. It remains to be seen how effective this information-intensive strategy will be at generating new clinically active agents.

Functional characterization of the S. cerevisiae genome by gene deletion and parallel analysis.

The functions of many open reading frames (ORFs) identified in genome-sequencing projects are unknown. New, whole-genome approaches are required to systematically determine their function. A total of 6925 Saccharomyces cerevisiae strains were constructed, by a high-throughput strategy, each with a precise deletion of one of 2026 ORFs (more than one-third of the ORFs in the genome). Of the deleted ORFs, 17 percent were essential for viability in rich medium. The phenotypes of more than 500 deletion strains were assayed in parallel. Of the deletion strains, 40 percent showed quantitative growth defects in either rich or minimal medium.

E2F and its developmental regulation in Xenopus laevis.

The transcription factor E2F has been implicated in cell cycle control by virtue of its association with cyclins, cyclin-dependent kinases, and pRb-related tumor suppressor gene products. Eggs and embryos from the frog Xenopus laevis have been used to investigate the characteristics of E2F-like molecules in the Xenopus cell cycle and throughout early development. We find multiple E2F species in Xenopus eggs, at least one of which is modified by phosphorylation. The vast majority of E2F remains in the free form throughout the very early embryonic cell cycle, and it also remains predominantly free until some time after the mid-blastula transition, the onset of zygotic transcription. At this time, E2F complexes significantly to pRb but not to cdk2, although cdk2 binding is found in tissue culture cells from a very advanced stage in embryogenesis. This suggests that the complexing of E2F to cyclins, cyclin-dependent kinases, and tumor suppressor gene products may be controlled separately in early Xenopus development. Thus, the association of E2F with other molecules may not result solely from processes affecting cell cycle progression but may also reflect developmental and differentiation cues.

Expression profiling using microarrays fabricated by an ink-jet oligonucleotide synthesizer.

We describe a flexible system for gene expression profiling using arrays of tens of thousands of oligonucleotides synthesized in situ by an ink-jet printing method employing standard phosphoramidite chemistry. We have characterized the dependence of hybridization specificity and sensitivity on parameters including oligonucleotide length, hybridization stringency, sequence identity, sample abundance, and sample preparation method. We find that 60-mer oligonucleotides reliably detect transcript ratios at one copy per cell in complex biological samples, and that ink-jet arrays are compatible with several different sample amplification and labeling techniques. Furthermore, results using only a single carefully selected oligonucleotide per gene correlate closely with those obtained using complementary DNA (cDNA) arrays. Most of the genes for which measurements differ are members of gene families that can only be distinguished by oligonucleotides. Because different oligonucleotide sequences can be specified for each array, we anticipate that ink-jet oligonucleotide array technology will be useful in a wide variety of DNA microarray applications.

Equal transcription of wild-type and mutant p53 using bicistronic vectors results in the wild-type phenotype.

Somatic and germ-line mutations of p53 alleles inactivate the function of the protein. It has been suggested that mutant p53 can inactivate the wild-type protein and therefore have a trans-dominant negative effect. To investigate the interaction between wild-type and mutant proteins when both alleles are equally transcribed, we designed bicistronic vectors containing the internal ribosome entry site of the encephalomyocarditis virus and expressing wild-type and mutant p53. Analysis of the transcriptional activity and of the effect on cell growth of these plasmids indicates that the mutant protein is unable to completely suppress wild-type function. These results could explain why the inactivation of both p53 alleles is required in cancer development.

A functional screen for germ line p53 mutations based on transcriptional activation.

Germ line p53 mutations represent a genetic predisposition for cancer development. At the present time, their detection requires extensive work and their functional significance must be documented. Therefore, we have designed a simple biological assay which detects functionally significant germ line p53 mutations. This assay is based on the cloning of the patient's p53 complementary DNA into a eukaryotic expression vector followed by the cotransfection into human cells of the recombinant vector with a reporter plasmid for the transcriptional activity of p53. This assay potentially offers a powerful method to screen fibroblasts or lymphocytes from patients for germ line mutations which inactivate the p53 tumor suppressor gene.

An abnormality in the p53 pathway following gamma-irradiation in many wild-type p53 human melanoma lines.

DNA-damaging agents such as ionizing radiation (IR) activate the tumor suppressor p53, and, in turn, p53 transactivates a number of downstream effector genes such as GADD45, CIP1/WAF1, and MDM2. The induction of these downstream genes following IR appears to be strictly dependent upon the presence of wild-type functional p53 known to evoke G1 arrest. In this study, we characterized 56 cell lines from 9 different tumor types with predetermined p53 genotype by measuring the induction of GADD45, CIP1/WAF1, and MDM2 relative mRNA levels after IR. A higher fraction of melanoma lines had wild-type (wt) p53 (5/8, or 63%) compared to the nonmelanoma lines (11/48, or 23%). Most wt p53 (nonmelanoma) cell lines (11/12, or 92%) showed clear induction of both GADD45 and CIP1/WAF1. On the other hand, many wt p53 melanoma lines (4/5, or 80%) showed normal induction of CIP1/WAF1, but little or no induction of GADD45. Despite this defect in GADD45 induction, we found that all wt p53 melanoma lines exhibited strong G1 arrest and increased levels of p53 protein after IR. The results demonstrated that radiation-induced G1 arrest could occur by the p53-CIP1/WAF1 pathway without appreciable induction of GADD45 in melanoma lines. Time course experiments demonstrated prolonged induced expression of CIP1/WAF1 mRNA transcripts in melanoma lines in which GADD45 induction was lacking, suggesting some sort of compensatory mechanism involving CIP1/WAF1, in cell lines with defective GADD45 induction. We could reproduce this compensatory effect in RKO colon carcinoma cells in which GADD45 expression was blocked by constitutive antisense vectors. These findings reveal that defective induction of GADD45 following IR is common in human melanoma cell lines.

Differential toxicities of anticancer agents among DNA repair and checkpoint mutants of Saccharomyces cerevisiae.

Most cytotoxic anticancer agents damage DNA directly, interfere with DNA metabolism or chromosome segregation, and are particularly toxic in dividing cells. Although a considerable amount of information on the mechanisms of action of these agents is available, the molecular bases for selective tumor cell killing by chemotherapy are largely unknown. Many genetic alterations found in sporadic and hereditary cancers affect functions in DNA repair and cell cycle control and result in sensitivity to DNA damaging agents. We have therefore set out to determine the effects of these cancer mutations on sensitivity or resistance to various chemotherapeutic agents. Because most of the affected genes are well conserved among eukaryotes, we have carried out a comprehensive analysis of a panel of isogenic yeast strains, each defective in a particular DNA repair or cell cycle checkpoint function, for sensitivity to the Food and Drug Administration-approved cytotoxic anticancer agents. Widely different toxicity profiles were observed for 23 agents and X-rays, indicating that the type of DNA repair and cell cycle checkpoint mutations in individual tumors could strongly influence the outcome of a particular chemotherapeutic regimen.