Effect of BRCA1 and XPG mutations on treatment response to trabectedin and pegylated liposomal doxorubicin in patients with advanced ovarian cancer: exploratory analysis of the phase 3 OVA-301 study.

BACKGROUND: We investigated the association of BRCA1 and XPG mutations with response rate (RR), progression-free survival (PFS) and overall survival (OS) in a subset of patients from a phase 3 clinical trial comparing the efficacy and safety of trabectedin + pegylated liposomal doxorubicin (PLD) versus PLD alone in patients with recurrent ovarian cancer. PATIENTS AND METHODS: A candidate array was designed based on the Breast Cancer Information Core database for BRCA mutation analyses. An exploratory analysis of BRCA1/XPG mutation status was conducted using a two-sided log-rank test and 0.05 significance in germline DNA samples from 264 women with failed first-line platinum-based chemotherapy, randomized (1 : 1) to trabectedin + PLD or PLD alone. RESULTS: Overall, 41 (16%) of the 264 women had BRCA1(mut) (trabectedin + PLD: n = 24/135, 18%; PLD: n = 17/129; 13%) and 17 (6%) had XPG(mut) (trabectedin + PLD: n = 8/135, 6%; PLD: n = 9/129, 7%). A higher RR was observed in BRCA1(mut) patients (20/41; 49%) versus BRCA1(wt) patients (62/223; 28%). Within the BRCA1(mut) group, trabectedin + PLD-treated patients had longer PFS and longer OS than PLD-treated patients (median PFS 13.5 versus 5.5 months, P = 0.0002; median OS 23.8 versus 12.5 months, P = 0.0086), whereas in BRCA1(wt) patients, OS was not significantly different (median OS: 19.1 versus 19.3 months; P = 0.9377). There were no differences in OS or PFS of patients with XPG(mut) between the two treatment arms. However, trabectedin + PLD-treated patients with XPG(mut) had a trend toward shorter PFS (median PFS: 1.9 versus 7.5 months; P = 0.1666) and OS (median OS: 14.5 versus 20.7 months; P = 0.1774) than those with XPG(wt). CONCLUSIONS: In this exploratory analysis, patients with recurrent ovarian cancer carrying the BRCA1(mut) had improved outcomes with trabectedin + PLD treatment compared with PLD alone. Prospective evaluation of BRCA status is likely an important evaluation for DNA-damaging agents and may significantly impact interpretation of clinical studies. XPG may be a biomarker of poor outcome in these patients.

Designing pharmacogenetic projects in industry: practical design perspectives from the Industry Pharmacogenomics Working Group.

Pharmacogenetic association studies have the potential to identify variations in DNA sequence which impact drug response. Identifying these DNA variants can help to explain interindividual variability in drug response; this is the first step in personalizing dosing and treatment regimes to a patient's needs. There are many intricacies in the design and analysis of pharmacogenetic association studies, including having adequate power, selecting proper endpoints, detecting and correcting the effects of population stratification, modeling genetic and nongenetic covariates accurately, and validating the results. At this point there are no formal guidelines on the design and analysis of pharmacogenetic studies. The Industry Pharmacogenomics Working Group has initiated discussions regarding potential guidelines for pharmacogenetic study design and analyses (http://i-pwg.org) and the results from these discussions are presented in this paper.

Classification of BRCA1 missense variants of unknown clinical significance.

BACKGROUND: BRCA1 is a tumour suppressor with pleiotropic actions. Germline mutations in BRCA1 are responsible for a large proportion of breast-ovarian cancer families. Several missense variants have been identified throughout the gene but because of lack of information about their impact on the function of BRCA1, predictive testing is not always informative. Classification of missense variants into deleterious/high risk or neutral/low clinical significance is essential to identify individuals at risk. OBJECTIVE: To investigate a panel of missense variants. METHODS AND RESULTS: The panel was investigated in a comprehensive framework that included (1) a functional assay based on transcription activation; (2) segregation analysis and a method of using incomplete pedigree data to calculate the odds of causality; (3) a method based on interspecific sequence variation. It was shown that the transcriptional activation assay could be used as a test to characterise mutations in the carboxy-terminus region of BRCA1 encompassing residues 1396-1863. Thirteen missense variants (H1402Y, L1407P, H1421Y, S1512I, M1628T, M1628V, T1685I, G1706A, T1720A, A1752P, G1788V, V1809F, and W1837R) were specifically investigated. CONCLUSIONS: While individual classification schemes for BRCA1 alleles still present limitations, a combination of several methods provides a more powerful way of identifying variants that are causally linked to a high risk of breast and ovarian cancer. The framework presented here brings these variants nearer to clinical applicability.

Detecting colorectal cancer in stool with the use of multiple genetic targets.

BACKGROUND: Colorectal cancer cells are shed into the stool, providing a potential means for the early detection of the disease using noninvasive approaches. Our goal was to develop reliable, specific molecular genetic tests for the detection of colorectal cancer in stool samples. METHODS: Stool DNA was isolated from paired stools and primary tumor samples from 51 colorectal cancer patients. Three genetic targets-TP53, BAT26, and K-RAS-were used to detect tumor-associated mutations in the stool prior to or without regard to the molecular analyses of the paired tumors. TP53 gene mutations were detected with a mismatch-ligation assay that detects nine common p53 gene mutations. Deletions within the BAT26 locus were detected by a modified solid-phase minisequencing method. Mutations in codons 12 and 13 of K-RAS were detected with a digital polymerase chain reaction-based method. RESULTS: TP53 gene mutations were detected in the tumor DNA of 30 patients, all of whom had the identical TP53 mutation in their stools. Tumors from three patients contained a noninherited deletion at the BAT26 locus, and the same alterations were identified in these patients' stool specimens. Nineteen of 50 tumors tested had a K-RAS mutation; identical mutations were detected in the paired stool DNA samples from eight patients. In no case was a mutation found in stool that was not also present in the primary tumor. Thus, the three genetic markers together detected 36 (71%) of 51 patients (95% confidence interval [CI] = 56% to 83%) with colorectal cancer and 36 (92%) of 39 patients (95% CI = 79% to 98%) whose tumors had an alteration. CONCLUSION: We were able to detect the majority of colorectal cancers by analyzing stool DNA for just three genetic markers. Additional work is needed to determine the specificity of these genetic tests for detecting colorectal neoplasia in asymptomatic patients and to more precisely estimate the prevalence of the mutations and sensitivity of the assay.

Universal DNA array detection of small insertions and deletions in BRCA1 and BRCA2.

Array-based mutation detection methodology typically relies on direct hybridization of the fluorescently labeled query sequence to surface-bound oligonucleotide probes. These probes contain either small sequence variations or perfect-match sequence. The intensity of fluorescence bound to each oligonucleotide probe is intended to reveal which sequence is perfectly complementary to the query sequence. However, these approaches have not always been successful, especially for detection of small frameshift mutations. Here we describe a multiplex assay to detect small insertions and deletions by using a modified PCR to evenly amplify each amplicon (PCR/PCR), followed by ligase detection reaction (LDR). Mutations were identified by screening reaction products with a universal DNA microarray, which uncouples mutation detection from array hybridization and provides for high sensitivity. Using the three BRCA1 and BRCA2 founder mutations in the Ashkenazi Jewish population (BRCA1 185delAG; BRCA1 5382insC; BRCA2 6174delT) as a model system, the assay readily detected these mutations in multiplexed reactions. Our results demonstrate that universal microarray analysis of PCR/PCR/LDR products permits rapid identification of small insertion and deletion mutations in the context of both clinical diagnosis and population studies.

Mutation detection in K-ras, BRCA1, BRCA2, and p53 using PCR/LDR and a universal DNA microarray.

We have developed a multiplex PCR/ligase detection reaction (PCR/LDR) that combines high sensitivity with the ability to simultaneously detect hundreds of mutations in a single-tube reaction. To enable us to rapidly assay large numbers of samples, we have linked this mutation detection scheme with analysis on a Universal DNA microarray. We have successfully applied this approach to characterize K-ras and p53 mutations in DNA derived from undissected colon tumors. The sensitivity of the assay has also facilitated detection of low-frequency mutations in BRCA1 and BRCA2 in pooled samples of DNA; thus, PCR/LDR can rapidly screen large numbers of DNA samples required for population studies.

5'-Nucleotidase in Dictyostelium: protein purification, cloning, and developmental expression.

5'-Nucleotidase (5NU) in Dictyostelium discoideum is an enzyme that shows high substrate specificity to 5'-AMP. The enzyme has received considerable attention in the past because of the critical role played by cyclic AMP in cell differentiation in this organism. Degradation of cAMP by cAMP phosphodiesterase (PDE) produces 5'-AMP, the substrate of 5NU. During the time course of development, the enzyme activity of 5NU increases and becomes restricted to a narrow band of cells that form the interface between the prestalk/prespore zones. We have purified a polypeptide associated with 5NU enzyme activity. Protein sequence of this peptide was obtained from mass spectrometry and Edman degradation. Polymerase chain reaction PCR amplification of genomic DNA using degenerate oligonucleotides and a search of sequences of a cDNA project yielded DNA fragments with sequence corresponding to the peptide sequence of 5NU. In addition, a clone was found that corresponded to the classical 'alkaline phosphatase' (AP) as described in several organisms. The sequences of the 5NU and AP cDNAs were not similar, indicating they are the products of separate genes and that both genes exist in Dictyostelium. Analysis of the expression of 5nu during Dictyostelium development by Northern blotting determined that the gene is developmentally regulated. Southern blot analysis showed a single form of the 5nu gene. Targeted gene disruption and knockout mutagenesis using the 5nu sequences suggested that a 5nu mutation may be lethal.

Transcription of the Dictyostelium glycogen phosphorylase-2 gene is induced by three large promoter domains.

The promoter of the Dictyostelium glycogen phosphorylase-2 (gp2) gene possesses a profound AT-bias, typical of promoters in this organism. To understand how Dictyostelium achieves specificity during transcriptional regulation under the constraint of this highly biased nucleotide composition, we have documented the changes in chromatin structure associated with developmental induction of gp2 gene expression. DNase I hypersensitive analyses indicated the presence of several developmentally regulated nuclease-sensitive sites located upstream of the start codon: two strong sites at approximately -250 bp and -350 bp and three substantially weaker sites at -290 bp, -445 bp, and -505 bp. In vitro footprint analyses using nuclear extracts derived from several stages of development (corresponding to varying levels of gp2 expression) revealed three large regions of occupation that were developmentally regulated and corresponded to these nuclease-sensitive sites: -227 to -294 bp (domain 1), -327 to -383 bp (domain 2), and -416 to -534 bp (domain 3). The presence and the extent of the three regulatory domains was confirmed by in vivo footprint analyses spanning the same developmental time points. Southwestern analyses using probes encompassing these footprints demonstrated that probes corresponding to domains 1 and 3 both interacted with 83 and 77 kDa peptides. The domain 3 probe also interacted with a 92 kDa peptide, while only a 62 kDa peptide is recognized by the domain 2 probe. In all cases, peptides capable of binding these probes were found in nuclear extracts derived from differentiated cells and not in undifferentiated cell nuclear extract. Using nuclear extract from differentiated cells and probes corresponding to the three domains, gel mobility shift analyses detected ladders of retarded bands for both domains 1 and 3 and three major retarded bands for domain 2. These results suggest that specificity in transcriptional activation in the AT-rich promoters of Dictyostelium may be achieved by requiring multiple protein-DNA and/or protein-protein interactions to occur before induction can proceed.

Isolation and characterization of glycogen synthase in Dictyostelium discoideum.

We have partially purified the protein and isolated the glcS gene for glycogen synthase in Dictyostelium. glcS mRNA is present throughout development and is the product of a single gene coding for 775 amino acids, with a predicted molecular mass of 87 kD. The sequence is highly similar to glycogen synthase from human muscle, yeast, and rat liver, diverging significantly only at the amino and carboxy termini. Phosphorylation and UDPG binding sites are conserved, with K(m) values for UDPG being comparable to those determined for other organisms, but in vitro phosphorylation failing to convert between the G6P-dependent (D) and -independent (I) forms. Enzyme activity is relatively constant throughout the life cycle: the I form of the enzyme isolates with the soluble fraction in amoebae, switches to the D form, becomes pellet-associated during early development, and finally reverts during late development to the I form, which again localizes to the soluble fraction. Deletion analysis of the promoter reveals a GC-rich element which, when deleted, abolishes expression of glcS.