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Q&A: Hanns-Georg Klein on Adopting Next-Gen Sequencing in a Clinical Diagnostics Lab


KleinPhoto2.jpgName: Hanns-Georg Klein
Age: 53
Position: CEO and clinical pathologist and medical geneticist, Center for Human Genetics and Laboratory Medicine Dr. Klein, Dr. Rost and colleagues (since 1998) and CEO, IMGM Laboratories (since 2001), Martinsried/Munich
Experience and Education:
Co-founder, Medigenomix (now part of Eurofins), 1997
Head of research group, Institute of Clinical Chemistry, University Hospital Großhadern, Munich, 1993-1998
Postdoctoral fellow, Molecular Disease Branch, National Heart, Lung, and Blood Institute, US, 1990-1993
Postdoctoral fellow and clinical training, Ludwig Maximilians University Munich, 1986-1990
MD, Ludwig Maximilians University Munich and Friedrich Alexander University Erlangen, 1986

The Center for Human Genetics and Laboratory Medicine Dr. Klein and Dr. Rost was founded in 1998 as a spinoff from the Institute of Clinical Chemistry at the University Hospital Großhadern in Munich.

With a staff of 90, about a third of them scientists or medical doctors, the privately owned center specializes in genetic and immunological diagnostics and analyzes about 30,000 samples per year, mainly from patients in Germany and Austria.

Diagnostic services include next-generation sequencing on the Roche/454 Life Sciences platform and the center is currently evaluating the Ion Torrent PGM and Illumina MiSeq instruments.

In 2001, the center founded a daughter company, IMGM Laboratories, that provides a variety of genomic services to researchers from industry and academia. Both firms share a building in Martinsried near Munich.

Clinical Sequencing News recently visited Hanns-Georg Klein, the center's founder and CEO, at his Martinsried office and talked with him about the advent of next-generation sequencing in clinical genetics. Below is an edited version of the conversation.

What kinds of molecular technologies do you use in your testing, and what role does next-gen sequencing play at the moment?

We provide classical cytogenetics, including FISH testing and hematological morphology. We use different platforms for array analysis, mainly for molecular karyotyping, either by array CGH or by SNP arrays. Of course we also use real-time PCR, like TaqMan or LightCycler assays. Sanger sequencing is still the gold standard for resequencing at the moment.

In terms of next-generation sequencing, we are most advanced in applying the 454 technology from Roche for HLA high-resolution sequenced-based typing and also for leukemia diagnostics, in particular for markers like the BCR-ABL fusion protein, where mutations in the gene cause resistance against tyrosine kinase inhibitors.

We also apply next-gen sequencing to the analysis of solid tumors, for example non-small-cell lung cancer, which is the most frequent sub-entity of lung cancer, and we analyze oncogenes like EGFR or KRAS and identify mutations which in turn enable specific therapy with antibodies, or, again, tyrosine kinase inhibitors, depending on the mutation status.

The main advantage of applying this technology is the higher sensitivity, because due to the clonal approach and the options for upregulating the coverage, we are able to detect minorities against the wildtype background of up to approximately 10 percent, which is far better than with Sanger sequencing, where we already run into problems with 30 percent to 40 percent minority [mutations].

We think we have a leading position worldwide in HLA sequencing by the 454 technology, which we have been offering for two years. The main advantage over conventional methods is cost reduction. We use the 454 because of the read length. It really provides an advantage over the short-read platforms because in HLA [typing], we have a highly variable germline template but we need haplotype information. The longer the read length, the easier the assembly work and the better the information on the haplotype.

What diagnostic applications are still based on Sanger sequencing today?

In fact, all the applications that have to do with resequencing of disease-associated genes in rare diseases. For example Marfan syndrome, familial cancer, breast cancer, or cardiac diseases, cardiac arrhythmias, cardiomyopathies. This is because at the moment, only Sanger sequencing is reimbursed by health insurance. The commissions on reimbursement are already working on specific reimbursement for next-generation sequencing, but at the moment, we don't have a definite concept yet on what kinds of rules and regulations might apply to next-generation sequencing for the diagnostic of rare diseases.

So there is no reimbursement for any NGS-based sequencing yet in Germany?

No. You would have to apply the regular reimbursement figure for sequencing, but the latest update of the physician scale determines that this can only be used when Sanger sequencing is applied, because obviously, with next-generation sequencing, you could multiply these [figures] by a thousandfold and therefore increase your revenues dramatically, which is not an acceptable situation for a budgeted system.

How is clinical next-gen sequencing regulated in Germany?

It's not regulated at all at the moment. Our group is currently in the process of submitting a manuscript to the Journal of Laboratory Medicine on quality standards of next-generation sequencing in diagnostics, which will probably be published in the summer issue.

What do you believe these quality standards should be?

We have to start with simple requirements, like minimum coverage, enrichment procedure recovery, and also for bioinformatics. The problem is that there are so many different platforms on the market available now. So far, we have only had Sanger sequencing, and with Applied Biosystems' capillary sequencing, there was more or less a worldwide unique method, highly standardized. Now we have at least three or four different platforms; in our laboratory, we are using or evaluating three platforms at the moment, the Roche/454, the Illumina MiSeq, and the Ion Torrent PGM.

It was difficult to define quality standards, but we will see what the feedback on our suggestions will be, and we have to continue discussing this. At the European Society of Human Genetics meeting in Nuremberg at the end of June, we will have workshops where we hopefully will further define these standards. Our goal is to have them defined by the end of the year, and to be able to get reimbursement by the beginning of next year.

Where do you see the greatest challenges for implementing next-generation sequencing in clinical diagnostics?

It's still a technical challenge; it's a much more complex workflow compared to Sanger sequencing. There are a lot of small steps that can go wrong. In two of the technologies we apply — the Roche/454 and the Ion Torrent — there is also a problem with homopolymer detection. And we still don't know how the systems perform in difficult regions, for example GC-rich regions or pseudogenes. The read length might play a role, though we don't know this yet.

I think the bioinformatics will also be challenging because we have to define which of the raw data we can actually use for the interpretation of the results. We also will have to be able to screen databases worldwide and compare them with our data in order to find out what the aberrations we find in our patients actually mean, what their clinical impact is.

And finally, the third challenge is to find a way to apply this powerful technology in a useful medical application that helps the patients. To do the research, getting more data and more information is one thing; but to translate this into information which is helpful for the patient is another matter. Imagine yourself getting your whole genome sequenced, and you get a result with 10,000 aberrations, of which only 1 percent are known. You might be restless until you have found out what's wrong with you, or what the other 99 percent of the variations mean that we cannot functionally explain at the moment. In other words, the technologies are probably more powerful than they have clinical utility at the moment.

How do you deal with findings of unknown significance?

We encounter this every day. As long as it is single variations we cannot interpret, we just provide it in our report as a non-interpretable variation. As long as there are one or two, I think it is no problem. But if you have a list of 30 or 50 or 100, it might create a problem for the patient, who is then becoming anxious about being at risk for a certain disease.

At the moment, we don't have any idea yet within the [German] Society of Human Genetics how we should deal with this unwanted information. My personal opinion is, we should release it, because I think that every patient has the right to get all the information. It might cause a lot of questions and consultations but I think that's the best way to deal with this information.

What about unintended findings that are interpretable?

There is also no clear opinion in the German community yet. I personally think if this variation causes a risk for the patient, we should tell him. The patient has to know that a genetic condition is not a 100 percent fate. There is always a chance that he doesn’t get the disease, or he might have other genetic factors protecting him from outbreak or alleviating the phenotype, and there is always a chance for new therapies.

If the patient has clearly stated in the informed consent that he doesn't want to have any additional information, even if it's deleterious for his health or his children or anyone related to him, then of course he won't get this information. But this is just my personal opinion; I'm not representing the German Society of Human Genetics.

Where do you see upcoming useful clinical applications of next-gen sequencing?

Our approach will be to define disease groups within a clinical entity that involve a limited number of genes. So this will be a targeted resequencing approach for most of the disease groups.

There is also already an application for whole-exome sequencing, particularly for mental retardation syndromes and malformation syndromes in children, which are genetically very heterogeneous.

I don't see, at the moment, a reason for whole-genome sequencing, which also involves, of course, the 95 percent of non-coding DNA. We know that there are regulatory elements and other things that might be important, but I think this is maybe the step after the next step.

Some laboratories may generally do whole-exome sequencing and then just do a bioinformatic pull-down of the data that were requested for the specific indication; others will only sequence those regions that are needed for the specific clinical question. We will probably try both approaches and then try to find our own approach.

Eventually, I also see an opportunity for next-gen sequencing in microbiology. With microbiology, we have some competition with mass spectrometry because this is also a method that is developing very fast. But I think next-generation sequencing will be able to identify complex spectra of germs and also be able to quantify them. Here, again, we will probably benefit from the high sensitivity in detecting minorities.

Of course research will be important, biomarker discovery. This will probably be a completely new field for diagnostic labs to get involved in, in collaboration with biotech and pharma companies, to identify genetic markers and biomarkers that will be used for companion diagnostics in the future.

How do you deal with the constant change in next-gen sequencing technology? Does that hinder the adoption of NGS for clinical applications?

We try to keep up with the constant change; we try to stay ahead. We will definitely not wait until we have the Swiss army knife of a sequencer because this will probably never exist. We were the company that brought diagnostic sequencing into routine use in Germany. This was our main achievement over the last 14 years. And we think we will also be the company that will bring next-generation sequencing on the market in Germany.

Is there anything else you'd like to mention?

I think that next-generation sequencing is really a quantum leap in terms of genetic diagnostics, comparable to the invention of the PCR technology. It's starting to be there; everybody is talking about it; some companies are already using it. We in our company have always been more conservative in releasing new technologies for routine use because everything we release undergoes rigorous internal validation. We are still in the validation process, but we think that it will dramatically change genetic diagnostics.

We believe that this tremendous information we receive based on these new technologies will require more clinical genetics and will also require more functional genetics, validation by other methods, for example biochemical tests or flow cytometry protein testing.

What we need to do is combine the information that is contained in the germline with what is actually happening in the organism, because not everything that is encoded in the germline really has an impact on the organism. We need to combine classical biochemistry with genetic information. And this will also be a quantum leap for patient information.

And finally, [next-gen sequencing] should be combined with imaging technologies, which have also made huge advances in the past. It's not only genetics where a lot of things are happening. That's why we think that expenses for diagnostics will increase from now maybe a share of 5 to 10 percent of total healthcare costs to up to 30 percent in the next 5 to 10 years. Because good diagnostics is a prerequisite for good treatment.