By identifying genes that are differentially expressed in carriers of BRCA1 and BRCA2 mutations, researchers in Israel are working on developing a test that, if validated, might offer a new approach to gauging people's risk of hereditary breast and ovarian cancer.
Furthermore, since this PCR-based test would gauge the expression of genes other than BRCA1 and BRCA2, the researchers, if they are successful, may have found a true workaround to Myriad Genetics' BRCA gene patents.
Researchers led by Asher Salmon of Hadassah Hebrew University Medical Center initially identified 21 genes that BRCA1/2 mutation carriers expressed significantly differently than non-carriers did. They then conducted a study to gauge the accuracy of these 21 genes, along with four housekeeping genes, in 40 BRCA 1/2 mutation carriers and 17 non-carriers by RT-PCR.
"RNA expression analysis of the 21 selected genes by RT-PCR revealed statistically significant differences between BRCA1 and BRCA2 mutation carriers and the control mutation-negative individuals," the study authors wrote in a paper published in Cancer Prevention Research in February. Then, the researchers narrowed the number of genes of interest down to 18 in an effort to develop a multiplex model that had the best sensitivity and specificity.
They ultimately settled on a model that included six or more coincidental genes of the 18 genes, yielding a positive predictive value of 95 percent and a negative predictive value of 88 percent to distinguish BRCA1 and BRCA2 carriers from non-carriers. Salmon and his colleagues cautioned in the paper that since the prevalence of BRCA1/2 mutations will be much lower in the general population than in the study population, the positive predictive value of the multiplex test will likely be lower.
The effort by Salmon and his colleagues to develop a gene expression PCR test evolved out of data from previous studies showing that patients with one copy of the mutated BRCA1 or BRCA2 gene have altered protein function, which causes a number of genes to be expressed differently than they are in those who don't have mutations in these genes. These altered genes are often linked to different biological functions, such as apoptosis, cell signaling, transcriptional activation, and DNA replication and repair processes, which play a part in the biological changes that lead to cancer.
"Alterations in the levels of BRCA1 and BRCA2 proteins in heterozygous cells might be expected to lead to multiple gene expression differences in breast and ovarian epithelial cells and in fibroblasts, suggesting … that BRCA heterozygosis itself can contribute to breast cancer initiation," Salmon and colleagues wrote in the paper. "Our results that mRNA expression profiles are altered following irradiation in BRCA1 or BRCA2 heterozygous cells compared with controls are also consistent with detectable heterozygous effects."
In order to identify the genes of interest, Salmon and colleagues collected fresh blood samples from known female BRCA1/2 carriers and non-carriers, who were between ages 25 and 50 years, with no personal history of cancer. The investigators identified women heterozygous for BRCA1/2 mutations and collected lymphocytes from their blood samples.
They then analyzed cDNA from lymphocytes from nine patients who were BRCA1 mutation carriers, eight carriers of BRCA2 mutations, and 10 healthy non-carriers using the Affymetrix GeneChip Human Genome U133A 2.0 Array. They conducted hybridization experiments on the samples before and after irradiation of the samples, which revealed that before irradiation the samples had no significant differences in gene expression profiles, but after irradiation gene expression patterns emerged between BRCA1/2 carriers and the control group.
Following this, the investigators tested samples from 27 mutation carriers and 12 non-carriers by RT-PCR in an effort to narrow down from 36 genes that were differentially expressed in the earlier investigation to 21 genes that were significantly differentially expressed. Salmon and colleagues further tested the accuracy of these 21 genes in samples from 40 BRCA1/2 carriers and 17 non-carriers, which yielded statistically significant differences in gene expression between the two groups.
Using statistical analysis, Salmon and colleagues were able to drill down even further to a set of 18 genes that were significantly downregulated in cells heterozygous for BRCA1 or BRCA1 mutations after irradiation. According to the study authors, half of these 18 genes have been associated with breast cancer in previous studies. For example, the STAT5 gene – one of the genes included in the multiplex model – has been shown to be inactivated in patients with breast cancer and has been associated with poor disease prognosis.
Following this latest study, Salmon and colleagues hope to conduct larger studies enrolling patients representing different populations. "We're planning one in Europe and another in North America … before we can go to the next step, which is basically commercializing [the test]," Salmon said.
He is hoping to develop the PCR test as a general screening tool for breast and ovarian cancer risk for the "developed market," as well as an adjuvant tool that healthcare providers can use when testing directly for BRCA mutations finds markers that have an uncertain association to cancer. Salmon estimates that between 10 percent and 15 percent of BRCA mutation tests yield variants of uncertain significance and may be "potentially positive" for a deleterious BRCA1/2 mutation.
Myriad Genetics, which markets the BRACAnalysis test that combines Sanger sequencing and PCR testing, currently holds a monopoly over BRCA mutation testing for hereditary breast and ovarian cancer risk. By enforcing its patents on isolated BRCA1/2 sequences, the company has been able to restrict other labs in the US from launching commercial tests that gauge cancer-linked mutations in these genes. Although the validity of several of Myriad's gene patents underlying BRACAnalysis test are currently being challenged in a high profile Supreme Court case (PGx Reporter 4/17/2013), the outcome of the lawsuit likely won't have much bearing on the ability of Salmon and his colleagues to launch their PCR test.
"Since we're looking into the expression patterns of other genes which coincide with elevated BRCA, it has nothing to do with the Myriad patents," Salmon told PGx Reporter. "And Myriad has never tried to challenge this issue. They have been quite aware for quite a while of my work."
Salmon and his colleagues suggest in the paper that a PCR test for determining which patients have BRCA1/2 mutations will offer certain advantages over available tests. "The available diagnostic tests for mutation analysis of BRCA1 and BRCA2 are time and labor intensive, expensive, and do not allow for the identification of all types of mutation (e.g., mutations in the regulatory regions, which are found up and downstream to the structure gene)," they wrote. The authors claim that their PCR method would gauge known and rare mutations in the structural and regulatory regions of the BRCA1/2 genes.
According to Salmon, a PCR-based test for hereditary breast and ovarian cancer risk will provide "a major price advantage" compared to BRACAnalysis and future next-generation sequencing-based tests. He estimated that the test he is developing would cost between $100 to $200. Comparatively, Myriad's BRACAnalysis costs between $3,000 and $4,000.