BERLIN (GenomeWeb) – Qiagen has been marketing its GeneReader next-generation sequencing system as a complete solution — with an automated sample prep, assay, and informatics workflow that includes instrumentation, reagents, and software for analysis and clinical reporting — to molecular pathology and diagnostics laboratories, initially for oncology applications.
At the Association for Molecular Pathology's AMP Global Congress in Berlin this week, several customers and users of the GeneReader presented results from initial studies conducted on the platform.
Unlike other vendors of next-gen sequencing systems, Qiagen charges labs for samples analyzed rather than for the platform itself. The price per sample depends on volume, a company representative said during a Qiagen-organized conference workshop, and is "very compatible with reimbursement codes in Germany and the US."
In addition, Qiagen does not disclose technical specs of the GeneReader sequencing instrument in writing, though a company representative said the sequencer generates mid- to high- single-digit gigabases of data per run across three flow cells.
The Medical University of Graz in Austria has been collaborating with Qiagen to develop new methods and workflows for analyzing molecular pathology biomarkers by NGS. The project is part of a program of the Center for Biomarker Research in Medicine (CBmed), which is funded by the Austrian government.
The university was the first institution outside of Qiagen to install the GeneReader system, according to Lisa Oberauner-Wappis, a postdoc in the Institute of Pathology who spoke during the workshop. She cited the workflow automation as an advantage, along with the fact that it includes several quality-control steps, a middleware software called GeneRead Link to transfer data between the GeneReader system and a LIMS, and protocols that can be accessed on a tablet computer.
Her team has been testing the GeneReader on samples from melanoma, lung, and colon cancer patients, including tissue samples from primary tumors and distant metastases, as well as blood samples taken before and after surgery. Allele frequencies of cancer mutations in these samples differed, ranging from 40 to 62 percent in the tissue samples and from 0.5 to 64 percent in the blood samples.
Specifically, she presented results from four melanoma patients, each with matched FFPE and liquid biopsy samples, that were analyzed using Qiagen's GeneRead QIAact Actionable Insights Tumor (AIT) panel, which covers hotspot mutations in 330 amplicons from 12 cancer genes.
Pathogenic variants detected in the matched samples from each patient largely overlapped, though the liquid biopsy assay detected additional variants in three cases, and the tissue assay in one case.
The group also compared matched FFPE and formalin-free PAXgene tissue-fixed paraffin-embedded (PFPE) samples from three colorectal cancer patients. Results for each of the three sample pairs were also largely concordant, with few variants called in one but not the other.
Overall, quality scores for the three sample types were high during the QC step, Oberauner-Wappis said, and all variants were correctly identified and interpreted, showing that the GeneReader can handle different types of samples reliably. Her team is now waiting to receive additional panels from Qiagen, including a lung cancer panel, a liver cancer panel, and a microbiome panel. In addition, she said, the group is planning to publish a study comparing results from the GeneReader and a Thermo Fisher Scientific Ion Torrent sequencer for a number of clinical samples.
Pangaea Oncology, a company based in Barcelona, Spain that is active both in patient hospital care and in molecular diagnostics, has also been testing the GeneReader for detecting cancer mutations in tissue and liquid biopsy samples.
Clara Maya de las Casas, director of Pangaea's liquid biopsy unit, said during the workshop that the company’s long-term aim is to implement liquid biopsy assays for cancer patients routinely, both at the time of diagnosis and at the time they progress, though cfDNA assays will likely never replace tissue-based assays for making diagnoses.
One of the challenges of liquid biopsy testing is that the amount of circulating free DNA can be very low, and that the fraction of circulating tumor DNA is often less than 1 percent of the total, she said, requiring highly sensitive and specific assay technologies. Also, for cancers such as lung cancer, many molecular alterations in several genes have been described that have targeted therapies associated, and it is important to be able to interrogate them in a single assay because there is often not sufficient material for multiple tests.
During the workshop, she presented some of the firm's work using the GeneReader, which the lab obtained last fall, for analyzing circulating free DNA from serum, plasma, and other body fluids. For its analytical performance test of the GeneReader, the lab tested 77 previously genotyped lung cancer samples using the AIT panel, including 64 matched plasma and serum samples from 32 patients and 13 other samples, including plasma, urine, cerebrospinal fluid, peritoneal lavage, and pleural fluid.
One problem was the low amount of DNA in many of the samples, she noted, which was often below the amount recommended by Qiagen, but despite this limitation, the lab obtained good results.
Only two samples failed sequencing, and for the remainder, 88 percent of results were fully concordant with previous genotyping results. In some of the discordant samples, the GeneReader detected one EGFR mutation but not another that was previously detected by genotyping at a very low frequency. Overall, results from matched serum and plasma samples agreed well, Maya de las Casas said, but plasma appeared to yield slightly better results.
She also presented results from a colorectal cancer patient who had progressed after therapy, where the GeneReader accurately reproduced multiple pathogenic variants in the KRAS and NRAS genes in the patient’s blood that the genotyping assay had previously identified.
In a blood sample from another case, a lung cancer patient with multiple disease progressions, the GeneReader detected a recently discovered pathogenic EGFR mutation that was on the AIT panel but outside the "region of interest" for interpretation, illustrating the importance of keeping panels and analyses up to date. Maya de las Casas noted that the mutation is included in a new lung cancer panel that Qiagen recently launched.
That assay, called GeneRead QIAact Lung DNA Panel, also uses a new unique molecular index (UMI) technology that barcodes each DNA molecule in the sample prior to amplification, allowing the assay to distinguish real mutations from errors introduced during PCR. The panel detects SNPs, indels, copy number variants, and gene fusions in 19 genes related to lung cancer.
In addition to the workshop reports, two GeneReader users, who both have the system installed at their site, presented posters at the conference. A group led by Helen Fernandes in the Department of Pathology at Weill Cornell Medicine, for example, tested the system’s performance for detecting mutations in FFPE tissue sections from thyroid, colon, lung, melanoma, and colorectal cancer, comparing it against its existing Ion Torrent PGM workflow.
Specifically, they used the GeneReader and the AIT panel to analyze 59 FFPE samples, 52 of which had known clinically relevant mutations with allele frequencies ranging from 2 percent to 88 percent. They also ran a number of samples with mutations in BRAF, KRAS, NRAS, and EGFR in triplicate to test reproducibility, which is recommended by guidelines for somatic variant detection from the New York State Department of Health.
The researchers were able to confirm all variants in the 52 samples with known mutations, and the variants were confirmed in all triplicate samples. Allele frequencies between the GeneReader and the PGM agreed well for KRAS, NRAS, and most BRAF variants but less so for EGFR variants, which they said might be due to the panel design. Because of its fully integrated sample prep, quality control, and bioinformatics solutions, the GeneReader system "is well positioned for clinical laboratories desiring to implement the NGS technology for routine analysis," they concluded.
Another study, led by Florian Haller and Jan Seitz at the Institute of Pathology at the University of Erlangen-Nuremberg, explored the GeneReader system for EGFR mutation testing in blood in patients with advanced EGFR-mutant non-small cell lung cancer.
They analyzed cfDNA from 13 NSCLC patients with known activating EGFR mutations in their primary tumor, using the GeneReader and the AIT panel. In six of the 13 cases, they were able to detect the EGFR mutation in the blood, with allele frequencies ranging from 0.6 percent to 8.5 percent. In addition, in one patient, they found a secondary EGFR mutation with an allele frequency of about 2 percent, which indicated that he had become resistant to therapy. Overall, the GeneReader system showed "sufficient sensitivity for further studies in the field of cfDNA diagnostics," the researchers wrote, and the results suggest that the approach "is of adequate diagnostic accuracy."