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University of Cologne Combines AmpliSeq, MiSeq for Cancer Dx; Looks to Validate Qiagen Panels

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The Institute of Pathology at the University of Cologne in Germany is now offering a lung cancer panel to guide treatment and enroll patients into clinical trials. The center has also been testing Qiagen's newly launched cancer panels for clinical use.

Lukas Heukamp, a pathologist at the University Hospital Cologne and co-lead of a larger lung cancer consortium in Cologne, told Clinical Sequencing News that the lab has developed and validated for clinical use a custom panel using Life Technologies' Ion AmpliSeq technology, which it runs on Illumina's MiSeq instrument.

Additionally, in evaluations of Qiagen's panels, also run on the MiSeq, results have been comparable to the AmpliSeq panels run on the MiSeq, he said.

While it was difficult to do a true head-to-head comparison, since Qiagen has fixed panels that target slightly different regions than the group's customized AmpliSeq panel, Heukamp said that looking at both "the [multiplex PCR] chemistry of the Qiagen panel and the primer panel of the AmpliSeq, the enzymes performed comparably well."

Currently, Qiagen markets a comprehensive cancer panel targeting 124 genes, as well as eight subpanels targeting relevant genes for breast, liver, lung, prostate, colon, gastric, and ovarian tumors, as well as leukemia (CSN 11/7/2012).

Recently, the company said that it plans to expand its number of fixed panels to 20 over the next several months. A company spokesperson told CSN that the panels will each cover between 300 and 9,500 mutations with a target region size of 4 kilobases to 96 kilobases and DNA input requirements between 20 nanograms and 80 nanograms.

Since Qiagen is currently only marketing fixed panels, Heukamp said he did not intend to switch from the lab's validated, customized panels for their clinical work, but he said that if Qiagen adds an element that enables customization, he would consider designing and validating a Qiagen panel for clinical use as well.

"We're not running the ready-made panels [for clinical use] because we are involved in a lot of Phase I and II trials in lung cancer, and the targets we need to sequence change quite rapidly and we often have new targets that are not included in the [fixed] panels," Heukamp said.

As such, the team has designed its own lung cancer panel using AmpliSeq chemistry, which includes 200 amplicons covering 15 genes and is a combination of full gene sequencing and hotspot sequencing.

For instance, he said, there is a clinical trial for which patients with mutations to the DDR2 gene are eligible. So the panel includes full sequencing of that gene. But in other genes, such as EGFR and BRAF, there are specific mutations that point to treatment options, so only certain regions of those genes are targeted.

Heukamp said that the researchers had originally created a panel of 600 amplicons targeting around 40 genes, but "realized we couldn't multiplex as many patients per sequencing run as we really wanted to." The panel also included "many genes that we were interested in but didn't really need," since they could not be linked to therapy or clinical trials at present.

He said he chose the AmpliSeq/MiSeq combination because at the time AmpliSeq was the only chemistry that could be customized. Additionally, it requires less input DNA than Illumina's TruSeq panel, which is important when dealing with cancer samples from formalin-fixed paraffin-embedded tissue, he said. The team went with MiSeq for the sequencing portion, however, because at the time he said the Ion Torrent PGM was still problematic when sequencing homopolymers, although he noted that Life Tech has been improving its performance in this area.

Heukamp said the center now has the capacity to sequence around 3,500 lung cancer patients per year using its customized panel. The University of Cologne is part of a larger network, the Network Genomic Medicine Lung Cancer, run by the Lung Cancer Group Cologne, of which Heukamp is also a co-PI. The Lung Cancer Group Cologne accepts FFPE samples from both university hospitals and small community hospitals in the region for sequencing.

"If patients have driving mutations for licensed therapies, they can continue to be treated in their local hospital," he said. "But if patients have a rare event that is eligible for a clinical trial at a cancer center, then they have the option to be enrolled here."

All patients with a primary diagnosis are eligible for the sequencing panel, Heukamp said. Currently, only EGFR testing is reimbursable, he added, so the center applies for reimbursement for testing that gene, and the remainder of the cost is covered with research money, so patients do not have to bear the cost of the test.

Aside from the lung cancer panel, Heukamp's team is also working on developing panels for gastrointestinal cancers and hematological malignancies.

For gastrointestinal cancers, including gastric, stomach, and colon, the team is not sequencing all-comers, but focusing on patients with later-stage disease. Those patients are most likely to benefit from the screening, since many of them, especially those with colorectal cancers, are successfully treated with surgery.

Additionally, in these cancers, translocations and copy number variations may play a larger role than point mutations, Heukamp said, so the sequencing panel may not be the primary focus.

Moving forward, Heukamp said that he would continue to evaluate Qiagen's sequencing panels and chemistry as the ability to customize is introduced. "If it works well, it's a really good option," he said, especially because Qiagen is designing its panels to be compatible with any sequencing technology. "It's an open system," he said, so "you're not fixed to a sequencing platform."

Qiagen is also planning to launch a next-gen sequencing system of its own for early access customers this year, which Heukamp said he would also be interested in testing (CSN 1/9/2013 and IS 2/26/2013).

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