NEW YORK (GenomeWeb) – A team of researchers from Germany has developed a targeted sequencing panel for chronic lymphocytic leukemia for use in diagnostics and clinical research.
The panel, which covers hotspot areas or complete coding regions of 15 genes known to be involved in CLL or associated with drug targets combines AmpliSeq multiplex PCR-based target amplification and sequencing on the Illumina MiSeq. Results from a proof-of-concept study involving 136 CLL patients and two mutated cell lines were published in PLOS One this week.
"Theoretically, the test would be extremely useful to monitor all CLL patients," Reinhard Büttner, one of the study's senior authors from the University Hospital Cologne in Germany, told GenomeWeb.
The Cologne team has validated the panel with a number of patient samples with known mutations and is currently using it in the clinic, especially to detect the emergence of resistance in CLL patients after treatment, he said.
The test is reimbursed by German statutory health insurance for inpatients only. For outpatients, reimbursement for next-generation sequencing tests by the German healthcare system has still not been established, he said, after it was halted in the fall of 2013.
Recent genome sequencing studies of CLL have discovered recurrent mutations in a number of genes, some of which appear to be associated with prognosis. According to the researchers, prospective clinical trials are now needed to develop prognostic or predictive biomarkers, and their panel could facilitate such research, as well as be used in diagnostics.
One advantage of next-gen sequencing for mutation analysis, they wrote in the article, is that it can pick up small subclones of cancer cells carrying mutations early, which can evolve over time and drive CLL progression.
For their panel, they selected genes known to be mutated in CLL — ATM, CD79B, DDX3X, FBXW7, MYD88, NOTCH1, SF3B1, TP53, and XPO — as well as genes involved in B-cell receptor signaling, some of which are drug targets: BTK, MAPK1, PIK3CA, PIK3CD, PTEN, and PTPN6.
They sequenced these genes in B-cell DNA extracted from blood samples of 136 CLL patients, most of them with untreated or early-stage disease.
To amplify the exons of the genes, they used custom AmpliSeq panels from Thermo Fisher Scientific, starting with 40 nanograms of B-cell DNA and running four separate multiplex PCR reactions. This was followed by sequencing on the Illumina MiSeq.
According to the researchers, the entire assay takes three days to perform, from DNA extraction to data analysis, which they wrote is "fast enough to be applicable to clinical decision processes."
Overall, 83 percent of the targeted exons were covered by at least 500 reads. For five exons only, the mean number of reads was below 100, insufficient for diagnostic purposes.
In 60 of the CLL patient samples, the researchers found at least one deleterious mutation in one of eight genes. Genes most frequently mutated were TP53, SF3B1, NOTCH1, ATM, and XPO1, and for those genes, the researchers tested for associations with clinical and prognostic parameters.
To estimate the assay's detection limit for mutations, they also sequenced two cell lines with known mutations that were mixed with wild type DNA in different ratios, and were able to detect a homozygous TP53 mutation in a background of 95 percent wild type DNA and a heterozygous ATM mutation in a background of 90 percent wild type DNA. "Thus, our method would be clearly able to pick up small mutated subclones in CLL, presumably beyond the detection limit of Sanger sequencing," they wrote.
"Our NGS methodology can be easily translated to molecular diagnostics of other types of cancer and may pave the way for a fast-throughput combination of morphological and molecular diagnostics in hematologic and non-hematologic malignancies," they concluded.
One challenge the researchers pointed out is being able to distinguish between somatic tumor mutations and germline mutations, especially in the absence of a normal control from the same patient, and they acknowledge that their list of mutations may contain variants that are not CLL-specific but are actually germline SNPs.
The team is not the first to develop a CLL-specific gene panel. Cancer diagnostics firm Cancer Genetics, for example, launched a CLL panel last year that targets actionable markers in seven genes.
Others are opting for more comprehensive panels. Foundation Medicine's FoundationOne Heme, for example, which targets patients with hematologic cancers, including leukemia, lymphoma, and myeloma, detects all classes of genomic alterations in 405 cancer-related genes.
Büttner said that because his team's assay is PCR-based, it is more sensitive to detect small subclones in a background of wild type cells than other tests, including minor clones that arise during drug resistance. "Usually the coverage with the [Foundation Medicine] test is limited," he said.
In addition, the assay is more comprehensive than many other CLL panels and covers "difficult" genes, such as NOTCH1.