Current Status of Companion and Complementary Diagnostics: Strategic Considerations for Development and Launch.

US Food and Drug Administration (FDA)-approved diagnostic assays play an increasingly common role in managing patients to prolong lifespan while also enhancing quality of life. Diagnostic assays can be essential for the safe and effective use of therapeutics (companion diagnostic), or may inform on improving the benefit/risk ratio without restricting drug access (complementary diagnostic). This tutorial reviews strategic considerations for drug and assay development resulting in FDA-approved companion or complementary diagnostic status.

Genome-wide association analysis implicates the involvement of eight loci with response to tocilizumab for the treatment of rheumatoid arthritis.

Rheumatoid arthritis (RA) is an immune-mediated inflammatory disease affecting the joints. A heterogeneous response to available therapies demonstrates the need to identify those patients likely to benefit from a particular therapy. Our objective was to identify genetic factors associated with response to tocilizumab, a humanized monoclonal antibody targeting the interleukin (IL)-6 receptor, recently approved for treating RA. We report the first genome-wide association study on the response to tocilizumab in 1683 subjects with RA from six clinical studies. Putative associations were identified with eight loci, previously unrecognized as linked to the IL-6 pathway or associated with RA risk. This study suggests that it is unlikely that a major genetic determinant of response exists, and it illustrates the complexity of performing genome-wide association scans in clinical trials.

A multicentre phase II gene expression profiling study of putative relationships between tumour biomarkers and clinical response with erlotinib in non-small-cell lung cancer.

BACKGROUND: Identification of appropriate markers for predicting clinical benefit with erlotinib in non-small-cell lung cancer (NSCLC) may be able to guide patient selection for treatment. This open-label, multicentre, phase II trial aimed to identify genes with potential use as biomarkers for clinical benefit from erlotinib therapy. METHODS: Adults with stage IIIb/IV NSCLC in whom one or more chemotherapy regimen had failed were treated with erlotinib (150 mg/day). Tumour biopsies were analysed using gene expression profiling with Affymetrix GeneChip microarrays. Differentially expressed genes were verified using quantitative RT-PCR (qRT-PCR). RESULTS: A total of 264 patients were enrolled in the study. Gene expression profiles found no statistically significant differentially expressed genes between patients with and without clinical benefit. In an exploratory analysis in responding versus nonresponding patients, three genes on chromosome 7 were expressed at higher levels in the responding group [epidermal growth factor receptor (EGFR), phosphoserine phosphatase (PSPH) and Rap guanine nucleotide exchange factor 5 (RAPGEF5)]. Independent quantification using qRT-PCR validated the association between EGFR and PSPH overexpression, but not RAPGEF5 overexpression, and clinical outcome. CONCLUSIONS: This study supports the use of erlotinib as an alternative to chemotherapy for patients with relapsed advanced NSCLC. Genetic amplification of the EGFR region of chromosome 7 may be associated with response to erlotinib therapy.

Association in multifactorial traits: how to deal with rare observations?

To detect the role of a candidate gene for a trait in a sample of individuals, we may test SNP haplotype or diplotype effects. For a limited sample size, many haplotype or diplotype categories may contain few individuals. This involves a power decrease when testing the association between the trait and the haplotypes or diplotypes as these categories provide little additional information while increasing the degrees of freedom. The present paper proposes a new strategy to group rare categories based on a measure of similarity between haplotypes or diplotypes and compares it to two other possible strategies to deal with rare categories: a SNP selection strategy based on haplotype diversity, and a grouping strategy that pools all rare categories into a single baseline group. This comparison is performed by means of simulation under four scenarios. We show that this new strategy shows the largest increase in power irrespective of the model underlying the candidate gene in the studied trait. This strategy therefore provides a powerful alternative to currently used methods to reduce the number of rare categories.

Improved use of SNP information to detect the role of genes.

A topical question in genetic association studies is the optimal use of the information provided by genotyped single-nucleotide polymorphisms (SNPs) in order to detect the role of a candidate gene in a multifactorial disease. We propose a strategy called "combination test" that tests the association between a quantitative trait and all possible phased combinations of various numbers of SNPs. We compare this strategy to two alternative strategies: the association test that considers each SNP separately, and a multilocus genotype-based test that considers the phased combination of all SNPs together. To compare these three tests, a quantitative trait was simulated under different models of correspondence between phenotype and genotype, including the extreme case when two SNPs interact with no marginal effects of each SNP. The genotypes were taken from a sample of 290 independent individuals genotyped for three genes with various number of SNPs (from 5-8 SNPs). The results show that the "combination test" is the only one able to detect the association when the two SNPs involved in disease susceptibility interact with no marginal effects. Interestingly, even in the case of a single etiological SNP, the "combination test" performed well. We apply the three tests to Genetic Analysis Workshop 12 (Almasy et al. [2001] Genet. Epidemiol. 21:332-338) simulated data, and show that although there was no interactions between the etiological SNPs, the "combination test" was preferable to the two other compared methods to detect the role of the candidate gene.

Genetic analysis of case/control data using estimated haplotype frequencies: application to APOE locus variation and Alzheimer's disease.

There is growing debate over the utility of multiple locus association analyses in the identification of genomic regions harboring sequence variants that influence common complex traits such as hypertension and diabetes. Much of this debate concerns the manner in which one can use the genotypic information from individuals gathered in simple sampling frameworks, such as the case/control designs, to actually assess the association between alleles in a particular genomic region and a trait. In this paper we describe methods for testing associations between estimated haplotype frequencies derived from multilocus genotype data and disease endpoints assuming a simple case/control sampling design. These proposed methods overcome the lack of phase information usually associated with samples of unrelated individuals and provide a comprehensive way of assessing the relationship between sequence or multiple-site variation and traits and diseases within populations. We applied the proposed methods in a study of the relationship between polymorphisms within the APOE gene region and Alzheimer's disease. Cases and controls for this study were collected from the United States and France. Our results confirm the known association between the APOE locus and Alzheimer's disease, even when the epsilon 4 polymorphism is not contained in the tested haplotypes. This suggests that, in certain situations, haplotype information and linkage disequilibrium-induced associations between polymorphic loci that neighbor loci harboring functional sequence variants can be exploited to identify disease-predisposing alleles in large, freely mixing populations via estimated haplotype frequency methods.

Marker segregation information in breast/ovarian cancer genetic counseling: is it still useful? Groupe Génétique et Cancer de la Fédération Nationale des Centres de Lutte Contre le Cancer.

The use of mutation screening of BRCA1 and BRCA2 genes as a genetic test is still to a certain extent limited and the oncogeneticist may want to use complementary approaches to identify at-risk individuals. In a series of 23 families with at least three breast or ovarian cancer cases, screened for mutations at BRCA1 and BRCA2 and typed for markers at both loci, we investigated the usefulness of marker segregation information at two levels: 1) to what extent can the indirect approach identify the mutation carrier status of screened cases and their first-degree relatives, and 2) in what way does it help to identify the gene implicated in a family in which neither BRCA1 nor BRCA2 mutation has been detected? Using the indirect approach, the carrier status of the screened case could be determined with quasi certainty in three families and with a high probability in eight families. This status could be inferred in unaffected first-degree relatives as almost certain in one family and as highly probable in six families. Fourteen mutations were found concurrently in our series. Among the nine mutation-negative families, we were able to conclude that a BRCA1 mutation most probably segregated in one and that a mutation other than BRCA1 and BRCA2 was probably involved in two families. Our results show that, in small families, little help is to be expected from linkage data and mutation screening is the only way of identifying the origin of a genetic predisposition in a family. Marker segregation information may be useful in some large breast/ovarian cancer families in which no BRCA1 or BRCA2 mutation has been detected.

BRCA1 sequence variations in 160 individuals referred to a breast/ovarian family cancer clinic. Institut Curie Breast Cancer Group.

An account of familial aggregation in breast/ovarian cancer has become possible with the identification of BRCA1 germ-line mutations. We evaluated, for 249 individuals registered with the Institut Curie in Paris, the prior probability that an individual carried a mutation that predisposes to these diseases. We chose 160 women for BRCA1 analysis: 103 with a family history of breast cancer and 57 with a family history of breast-ovarian cancer. To detect small mutations, we generated and analyzed 35 overlapping genomic PCR products that cover the coding portion of the gene, by using denaturing gradient gel electrophoresis. Thirty-eight truncating mutations (32 frameshifts, 4 nonsense mutations, and 2 splice variants) were observed in 15% of women with a family history of breast cancer only and in 40% of those with a history of breast-ovarian cancer. Twelve of 25 distinct truncating mutations identified were novel and unique. Most BRCA1 mutations that had been reported more than five times in the Breast Cancer Information Core were present in our series. One mutation (5149del4) observed in two apparently unrelated families most likely originates from a common ancestor. The position of truncating mutations did not significantly affect the ratio of the risk of breast cancer to that of ovarian cancer. In addition, 15 DNA variants (14 missense mutations and 1 neutral mutation) were identified, 9 of which were novel. Indirect evidence suggests that seven of these mutations are deleterious.

Integrated map of the chromosome 8p12-p21 region, a region involved in human cancers and Werner syndrome.

Detailed physical maps of the human genome are important resources for the identification and isolation of disease genes and for studying the structure and function of the genome. To improve the definition of the 8p12-p21 chromosomal region, an integrated physical and genetic map was constructed extending from the genes. NEFL to FGFR1. The map comprises a series of contigs (the larger of these being around 9 Mb) of yeast artificial chromosomes (YACs) spanning the proximal region of deletion involved in a broad range of human cancers, including breast carcinomas, and in the Werner syndrome. In addition, losses of heterozygosity at 8p markers and linkage analysis of breast cancer families were also detailed. Finally, several genes potentially involved in 8p-associated diseases, namely GTF2E2, PPP2CB, and HGL, were precisely mapped within the YAC contigs. The reported map and contigs of YACs should facilitate the search for putative genes involved in sporadic and familial breast cancer as well as in the Werner syndrome.

Genetic epidemiology of breast cancer: interest of survival analysis methods.

In an attempt to explain the controversy resulting from the analysis of the breast cancer data collected by Jacobsen, a segregation analysis was performed using successively the unified mixed model (UM) and the logistic hazard function model (LHM) (Abel & Bonney, 1990). Under the UM, age of onset of the disease cannot be taken into account, each individual being assigned to a liability class according to his age at examination, whereas, in the LHM, variable age of onset is modelled using survival analysis methods. Under the UM, we confirmed the results of Demenais et al. (1986b), i.e. the transmission probabilities are significantly different from Mendelian expectations. The same results were obtained when taking into account the specific mortality for the computation of the morbid risk observed in a given liability class. Under the LHM, the analysis provides evidence for a monogenic autosomal model with a rare dominant allele responsible for the disease, and transmission probabilities compatible with Mendelian expectations. This study shows that the rejection of the Mendelian transmission under the UM can be due to a violation of a constraint of this model (i.e. the probability of being and not being affected in a given liability class should sum to 1) when a specific mortality is induced by the disease as in breast cancer. Survival analysis methods avoid these problems by taking into account the onset of the disease as the failure time event and are more suitable when studying a complex trait such as breast cancer.

Loss of heterozygosity and linkage analysis in breast carcinoma: indication for a putative third susceptibility gene on the short arm of chromosome 8.

We have analysed losses of heterozygosity (LOH) at eight markers from the p12-p22 region of human chromosome 8 in a panel of 113 breast tumors. LOH were detected in almost half of the tumors. The most frequently deleted region included microsatellite (CA)n repeats markers D8S258, D8S133 and D8S259, located at 8p12-p22, while markers NEFL and LPL appeared less frequently altered. In parallel, linkage analysis was performed using the same informative markers, to test for the involvement of chromosome 8p loci in familial breast cancer. Positive cumulative multipoint lod score of 2.51 at theta = 0.0 was obtained with markers NEFL and D8S259. These results suggest that region 8p12-p22 carries at least one tumor suppressor gene involved in sporadic and perhaps also in familial breast cancer.

Genetic counselling in breast cancer: sensitivity to parameter values and to available information.

Most segregation analyses have concluded that breast cancer results from a mixture of sporadic and genetic cases. Genetic cases are probably due to a rare inherited mutation with autosomal dominant transmission. The lifetime risk for female mutation carriers is close to 1, ten fold greater than for noncarriers. Beyond these accepted results, studies differ in the estimated parameters specifying the correspondence between phenotypes and genotypes. We show here that these differences have an impact on the estimation of the probability that a woman is carrying a mutation given her disease status and also given the information on the disease status of her family members. We illustrate this problem by computing the probability of being a mutation carrier for 16 women (9 affected, 7 unaffected) belonging to one pedigree, using three sets of parameter values. For two of the women, the probability that they inherited the mutation is low, but it varies considerably according to the set of parameter values. For one woman, the probability varies from 20 to 60% if familial information is taken into account. Segregation of 17q markers in families may provide additional information depending on the posterior probability of linkage. Indeed in the pedigree studied here, the segregation of 17q markers provided additional information which moreover decreased the sensitivity to the parameters values. However, the decrease in sensitivity will only be observed if the posterior probability of linkage of the family to the studied markers is high.

Analyses of linkage to 17q11-q23 in 3 French hereditary nonpolyposis colon-cancer families.

Hereditary non-polyposis colorectal cancer (HNPCC) is an autosomal dominant disease, accounting for approximately 6% of colorectal cancers. We performed linkage analyses with the aim of proving or excluding the existence of a susceptibility locus on 17q. Three HNPCC families (102 collected members, 25 colorectal cancers, 9 other cancers and 6 colorectal adenomas) were studied with 7 polymorphic DNA markers Mfd15, THRA 1, D17S800, D17S855, Mfd 188, 42D6, 46E6 localized in the 17q11-q23 region. After in vitro enzymatic amplification, the different alleles were separated by classic vertical poly-acrylamide gel electrophoresis or analyzed with the automatic sequencing machine 373A (Applied Biosystems). Results showed that none of the 7 studied markers of the chromosome 17q were linked to the HNPCC disease.

Linkage analyses of 3 French families to Loci on chromosome-2p and chromosome-3p predisposing to hereditary nonpolyposis colon-cancer.

Hereditary, non-polyposis colon cancer (HNPCC) is caused by mutations in different loci. One gene causing HNPCC was mapped to chromosome 2p and recently a tight linkage between a polymorphic marker on the chromosome 3p and the disease locus has been demonstrated and these families also manifest signs of a general DNA replicator disorder. We report detailed genetic studies of three French HNPCC families with D2S123 and D3S1029. In one of the families (F 230), the segregation pattern for markers on chromosomes 2 and 3 suggests absence of linkage. The two other families are not informative enough to conclude on linkage status with chromosomes 2 and 3. If confirmed, this result would mean that the inherited colon cancer in this family is linked to another HNPCC gene. Implication for genetic counselling is discussed. Even with cloned genes, linkage analysis with flanking microsatellite markers for informative families may help to avoid tedious work of seeking point mutations in HNPCC genes.

The effect of population structure on the relationship between heterosis and heterozygosity at marker loci.

The relationship between heterozygosity at neutral marker loci and heterosis of F1 hybrids is investigated using a theoretical model. Results emphasize that linkage disequilibrium between the markers and the loci implicated in heterosis [quantitative trait loci (QTLs) that exhibit dominance effects] is a necessary condition to finding a correlation (ϱ mh ) between heterozygosity at marker loci and the heterosis. The effect of population structure, in which the parental inbred lines of the hybrids belong to different heterotic groups, is considered. ϱ mh is investigated for: (1) hybrids between lines that belong to the same heterotic group (within-group hybrids); (2) hybrids between lines that belong to different groups (between-group hybrids); and (3) all hybrids, both within and between-groups. Within a group, significant values of (ϱ mh ) may arise because of linkage disequilibrium generated by drift. At the between-group level, no correlation is expected since link-age disequilibrium should differ randomly from one group to the other, which is consistent with recent experimental results. Possible ways to achieve prediction of the heterosis in this situation are discussed. When all hybrids are considered simultaneously, divergence of allelic frequencies among groups for the markers and the QTLs produces a correlation between heterosis and heterozygosity at marker loci. This correlation increases with the number of markers that are considered.