CAP TODAY Pathology/Laboratory Medicine/Laboratory Management
William Check, PhD
January 2015—Molecular genetics laboratories in the U.S. are doing a great job.
Ten-year data from the molecular genetics Surveys in the CAP proficiency testing program show that U.S. clinical laboratories are making extremely accurate calls using molecular genetics assays.
At the 2014 meeting of the Association for Molecular Pathology, Karen E. Weck, MD, and Iris Schrijver, MD, presented results from seven of the proficiency Surveys that the CAP/ACMG Biochemical and Molecular Genetics Resource Committee oversees. Dr. Weck is the chair of the committee; Dr. Schrijver is past chair.
“We have seen excellent performance of molecular genetics testing in general,” Dr. Weck says. “Overall sensitivity has been greater than 95 percent and specificity greater than 99 percent for all analytes we have evaluated thus far.” (Analysis of females for Fragile X had a sensitivity of 95.5 percent; all other sensitivities were around 99 percent.)
“Laboratories are doing a good job across the board,” Dr. Schrijver tells CAP TODAY. The committee members selected seven Surveys for analysis ranging from commonly performed cystic fibrosis testing to complex Fragile X syndrome testing, which has multiple components. They published results from these analyses over the past two years in a series of articles in Genetics in Medicine.
Analyzing molecular genetics laboratory performance is important for two reasons, says Dr. Weck, director of molecular genetics and professor of pathology and laboratory medicine and of genetics at the University of North Carolina. First, it can inform the molecular genetics laboratory community what expectations should be in terms of different methods. Second, it helps to identify areas where efforts need to be made to improve testing, “such as standardization or calibration or guidelines for interpretation,” she says.
Surveys provide more than numbers, says Dr. Schrijver, director of the molecular pathology laboratory at Stanford University Medical Center and professor of pathology and of pediatrics, Stanford University School of Medicine. “It is important not just to look at the data in the Surveys, but to provide educational content. Laboratories have improved considerably over time, particularly in the interpretive analysis,” Dr. Schrijver says. “They’re learning from participating in the Surveys.”
Further, results of these analyses shed light on the performance of laboratory-developed tests. “Of the greater than 30 molecular genetics Surveys overseen by our committee, almost all are performed by LDTs,” Dr. Weck points out. All of the methods used in the seven Surveys analyzed were laboratory-developed tests. “There are no U.S. Food and Drug Administration–approved test platforms for most of these disorders,” Dr. Schrijver says. The data show molecular genetics laboratories to be highly accurate when using LDTs.
In addition to the CAP analyte-specific molecular genetics Surveys, methods-based PT for Sanger sequencing has been offered for several years, which provides a segue to PT for next-generation sequencing. The CAP is launching a methods-based PT program for NGS, with the first PT mailing to take place in March. “Proficiency testing for next-generation sequencing will definitely be more challenging,” Dr. Weck says.
Dr. Voelkerding
Karl V. Voelkerding, MD, is chair of the CAP Next-Generation Sequencing Project Team, which is responsible for developing laboratory accreditation requirements and proficiency testing for next-generation sequencing. “In 2013–2014 we conducted a pilot program to assess a potential proficiency testing program for next-generation sequencing,” says Dr. Voelkerding, professor of pathology at the University of Utah and medical director for genomics and bioinformatics at ARUP Laboratories. “The results of that pilot were sufficiently encouraging to move forward and launch an educational PT program in 2015. When we receive and review results from participating laboratories, they will be used to refine the proficiency testing program.” The first next-generation sequencing-based PT program will focus on the ability of laboratories to detect sequence variants in germline DNA.
Dr. Schrijver credits the late Jeffrey Kant, MD, PhD, for the decision to analyze Surveys in molecular genetics. “This was a vision that originated with Jeff Kant to document performance of laboratories on individual assays and to share that information publicly,” she says. When Dr. Schrijver was chair of the CAP/ACMG committee, she put that plan in motion. “We were not sure what we would find,” she says. “We went back as far as each Survey existed.” CAP staff did the basic analysis, first-level interpretations, and summaries for the committee. All data were anonymized.
There was no selection bias among laboratories, Dr. Schrijver says. “All diagnostic laboratories in the U.S. have to be CLIA-certified and accredited by some organization. Virtually all molecular genetic laboratories are accredited by the CAP.”
For the Fragile X syndrome Survey, DNA from Coriell cell lines with known FMR1 genotypes was distributed. Individual laboratory responses were analyzed for accuracy of genotype determination and triplet repeat size. Sizing of the CGG triplet repeat region can be extremely important in some size ranges.
Sensitivities for full mutations were 99 percent in males and 95.5 percent in females; sensitivity for premutation was 98 percent. Specificity was 99.9 percent (Weck KE, et al. Genet Med. 2012;14:306–312). “Interlaboratory sizing by PCR improved over time, coincident with an increase in use of capillary electrophoresis over gel-based PCR and the availability of well-characterized materials for calibration,” Dr. Weck said.
For Huntington disease, diagnosis depends on detection of CAG repeat size in the HTT gene. By this criterion, analytic sensitivity found in the Survey was 99.5 percent and analytic specificity was 99.2 percent (Palomaki GE, et al. Genet Med. 2012;14:69–75). Plotting performance of individual laboratories over time showed that U.S. laboratories performed significantly better than non-U.S. laboratories. (This was observed in a few other Surveys as well.) “International laboratories are not CLIA-certified,” Dr. Schrijver notes. “They might be research laboratories.”
Sensitivity and specificity for Ashkenazi Jewish founder mutations in the BRCA1 and BRCA2 genes were found to be 99 percent and 99.9 percent, respectively (Tafe LJ, et al. Genet Med. Published online June 19, 2014. doi:10.1038/gim.2014.77). Only an average of 23 U.S. laboratories participated per year in the Survey, most likely due to patent issues. “Now that the patent has lifted, we will probably see more laboratories participating,” Dr. Weck says. Another consequence of the patent lifting is that the committee plans to extend the Survey to full gene-sequence analysis. Even so, the three mutations that have historically been analyzed by the Survey account for 95 percent of pathogenic mutations in the Ashkenazi Jewish population.