Transcript

Welcome to AACC’s Expert Access Live Online program

Our topic this month is Revolutionizing Molecular Medicine

This month's expert is V. Randy White, PHD, currently CEO of Xenomics, Inc. View the presentation and direct your questions to our online expert. AACC would like to thank Bayer HealthCare Diagnostics for making this program possible.

what form does the circulating DNA take i.e. is it supercolied, commplexed with histones etc?
boston

V. Randy White: Tr-DNA appears to have the histone shell intact; this may be one of the reasons Tr-DNA is relatively stable in urine. Although nucleases are present in urine, Tr-DNA is stable for several days without preservative and indefinately with preservative.

what is maximum size of DNA fragments filtered through the kidney
boston

V. Randy White: The fragments are usually right around 150 base pairs although we sometimes see fragments as large as 200 base pairs.

What is the amount of fetal DNA you detect in the urine (deoending on gestational ages)?
Bristol, UK

V. Randy White: We do not know this for a large population of women yet. We are starting a study now that includes 400 pregnant women and urine samples collected will cover the gestational age across the entire spectrum of pregnancy including 48 and 72 hours after delivery. In this way we hope to plot the kinetics of fetal Tr-DNA appearance in the mother’s urine and disappearance after delivery. This study will tell us the gestational week at which sufficient fetal DNA is present across a wide spectrum of women that will allow us to reproducibly perform the testing.

This is from a lay person. With the PSA test falling into disrepute as an effective screening mechanism for detecting prostate cancer, what chance is there that Tr-DNA testing could become an effective prostatge cancer detection test?
Wauwatosa, WI

V. Randy White: PSA is a protein marker produced by the prostate. As shown in slide #29 Tr-DNA in urine is a very sensitive way to look for various cancer mutations such as k-ras and p-16 methylation. Although mutant k-ras is expressed in prostate cancer, it is also expressed in several other types of cancer, so by itself it is not an acceptable “screening” test. However, it may be an ultra-sensitive way to monitor post-surgical re-growth. Based on calculations we believe we can detect prostate cancer re-growth with as few as 1000 cells. This could become very important with the new so-call soft therapies and cancer vaccines that are effective only at the earliest stages of cancer.

what is basic transrenal analysis(introduction to )
nigeria

V. Randy White: It is the same as for any molecular test: extract DNA, amplify DNA and detect. There is some proprietary enabling technology associated with Tr-DNA but it fits within this general sequence of events. Obviously once you have isolated DNA it can then be taken forward with any existing amplification/detection scheme. It works with PCR, bDNA, SDA, Invader, etc.

Can this be used to detect vasculitis?
Albany, NY

V. Randy White: If you are talking about non-TB inflammation of the vessel, I doubt it, but I simply don’t know. If you know of specific DNA-based changes associated with vasculitis, then it is possible and we would like to talk with you.

I'm surprised that your patent covers such a broad area as DNA in urine. Extracting genetic material in this matrix is hardly a novel technique, so can you outline--without giving away any trade secrets--how your method differs from others?
Ann Arbor, MI

V. Randy White: Our method differs in that Tr-DNA originated “inside” the body and not from shed cells of the renal system. The very best example is the ability to interrogate DNA of fetal origin in a simple collection of the mother’s urine. As to extraction what could be a better starting material than DNA in a high-salt matrix? Remember Tr-DNA is low-molecular weight DNA, so by using a special molecular sieve it is easy to separate low molecular weight DNA from bulk DNA from shed cells (see slide #7). From there virtually any commercial extraction system can be used and that carries forward into the detection platform - - our technology works with any existing amplification/detection system which is one of our business strategies. Because of the strength of our IP and the very broad claims, Xenomics doesn’t intend to manufacture a detection platform. Instead, we intend to manufacture reagent sets that will work on existing platforms installed in the laboratories - - i.e. we intend to make razor blades for the razors already installed. This should ensure market adoption, because the laboratory doesn’t have to buy new capital equipment, re-train personnel, or interface a new platform into the data system.

Very nice presentation on what is likely to be the future of molecular medicine. What platforms do you foresee for these tests: microarrays, licensed sets of commercial primers, a multiplex approach? Please elaborate.
Lisbon, Portugal

V. Randy White: This is similar to the previous question, but deserves elaboration. Because Tr-DNA works with any amplification/detection platform, Xenomics intends to make different reagent sets that will work on selected existing platforms. This will allow us to develop strategic partnerships with selected partners than will include a variety of amplification/detection platforms. Perhaps more importantly is your reference to the future of molecular medicine. Because the entire genome is represented in the Tr-DNA fragments and because those fragments are 150 base pairs long (more than sufficient for any marker), it begs the question why would you ever need a blood sample? For laboratory workers the non-infectious nature of a urine sample is important. Not to be missed is the size of the sample. A typical random urine is about 70 ml. Right now we are using 3 ml to start. If we needed to increase our sensitivity 10-fold we could simply start with 30 ml. To do the same with blood would require drawing 10 tubes of blood.

Would this technology be applicable for use in detecting early onset organ damage - i.e. before significant elevations in serum markers would be evident?
St. Louis, MO

V. Randy White: If you are talking post transplant, the answer is most definitely yes. Further we think it would be very easy to do, because donor DNA is unique from recipient DNA. If you are talking organ damage such as liver damage from hemochromatosis, it would probably be easier and more sensitive to measure the markers for hemochromatosis.

Are you providing analysis of urine specimens for tr-DNA? How much does it cost?
Moreno Valley California

V. Randy White: No, we are a development stage company working to produce molecular diagnostic products in two areas: prenatal genetic testing and infectious disease. Our Down syndrome screening test is just starting the first phase of clinical trials as our HIV, TB and malaria tests. We expect our products will compete favorably with other molecular diagnostic tests and in certain cases like the Down syndrome test may command a premium because it provides a definitive result and is a “first-trimester” test.

Have you had any problems supressing PCR inhibitors? As you know, this is a major challenge in analysis of plasma DNA.
Dallas, TX

V. Randy White: No we have not. Interestingly, because urine is a pretty clean matrix, we recently tried a series of experiments where we took diluted urine directly into the PCR reaction. There was a noticeable inhibition, but we were surprised that we still got significant product. If fact, we are working with reaction conditions to see if there is a feasible approach to not extracting Tr-DNA, but simply taking a diluted urine sample directly into the reaction. It remains to be seen.

In your presentation, cancer applications of Tr-DNA are limited to tumor monitoring and disease progression, not diagnosis. Might this technology eventually be used in cancer detection when we have more information on the exact function and nature of relevant genetic tumor markers?
Aberdeen, Scotland

V. Randy White: Absolutely! We the scientific community do not know enough yet about the combination of markers that are “diagnostic” for a particular cancer. We do however know that certain markers are always associated with particular types of cancer. That at least gives us a basis for monitoring progression and re-growth. It also gives on one more important application that I did not discuss: Let’s take a cancer that is k-ras positive. Let’s also realize that not all tumors respond the same to the various chemotherapy agents. Xenomics technology holds the potential key to adjusting chemotherapy real-time to find the “right” chemotherapy for the particular patient. Today physicians put a patient on a chemotherapeutic agent and then re-examine the patient in 60 days to determine if the tumor got smaller, remained the same, or grew. In the latter two, they change therapeutic agents and repeat the process. If we know the tumor is k-ras positive, then potentially we can baseline the patient, give the chemotherapeutic agent an re-measure the patient in 48 hours. Those patients with spiking k-ras are responding to the drug, the others are not.

According to your last answer, how sure are you on whether histone shell is intact on Tr-DNA?
SF

V. Randy White: We are pretty sure. We've run experiments by spiking labeled DNA into uine and watching its degredation which is vastly different than Tr-DNA. We believe it is the histone shell that affords this protection, but we do not know for sure.

Is this product available in Europe, or is it only in the States?
London, UK

V. Randy White: It is not available yet anywhere. A few weeks ago we did have our first European patent allowed and expect more in the future. Our commercialization plans certainly include Europe as we believe it is a large market. In a recent press release, the Company announced that I would be speaking at the Frankfurt Stock Day on September 1st. This is our initial attempt to get broader recognition of the power and potential of Tr-DNA in Europe.

Forgive my ignorance, but how does fragment size affect isolation and analysis of DNA?
Washington, DC

V. Randy White: Fragment size doesn't effect isolation. But Tr-DNA is a small fragment of 150 bp. We work exclusively with Tr-DNA because that is the DNA that originated "inside" the body. In the case of fetal DNA that passes through the placenta into the mother's blood stream and then through her kidneys. You can isolate the total DNA in a urine sample, but then you are adding background with the buld DNA. Better to first seperate short fragment DNA from bulk DNA to lower background.

Hello, Dr. White. Great presentation and excellent concept. Who holds the patent for this discovery? Thanks.
Newark, NJ

V. Randy White: Dr. David Tomei and Dr. Samuil Umansky discovered Tr-DNA and they are both founders of Xenomics and have assigned the three issued patents to Xenomics. Unlike most start-up companies, our patents have issued in the U.S. and our first patent in Europe was recently allowed. Beyond the seminal core patents we have nine other patents pending.

It would seem that TrDNA is easier to detect as the tissue source increases in mass (e.g., a fetal source versus a small tumor). Do you have a feeling for how many dead cells are needed to undergoe apoptosis, per day, to have a detectable TrDNA signal in the urine?
San Diego, CA

V. Randy White: I think I mentioned in an earlier question that on the basis of our sensitivity and considering that a cell contains 6 pg of DNA we made a calculation and believe that we can detect tumor markers when as few as 1000 cells have formed. This is theoretical and not proven, but that is our equivalent sensitivity.

Are you aware of any studies that examine the effect of renal dysfunction on the quality and quantity of DNA excreted in urine? Perhaps you and your colleagues have looked at this?
New Orleans, LA

V. Randy White: Excellent question. No I am not aware of any studies, but it is certainly an important feature for Tr-DNA analysis. We have very limited and very little data on nephrotic diseased patients and it will have to be one of the studies conducted as we move forward in product development. Obviously a diseased kidney may significantly clear more DNA than a normal kidney. If fact, the mild hyperenwion experienced during pregnancy may be one of the contributing reasons that a significant amount of fetal DNA is present in mother's urine. These studies need to be conducted.

When do you anticipate this technology will become commercially available and what are the potential obstacles that could delay this ? Thank you
calgary canada

V. Randy White: We hope to be in a position to make a 510K filing with the FDA sometime in 2007 for our Down syndrom screening test. A lot of work still need to be done and one of the rate limiting steps is the absolute frequency of Down syndrome events. We've tried to enrich the population by working with high-risk pregnancy centers. Our two clinical partners had 150 events in 12 months. We are comparing against amniocentesis and "outcome" and are hopeful that by submitting triangle data points that the FDA will accept the test as a 510K. If they do not, then it will take another year to complete the PMA version.

Could you elaborate on detection of proviral HIV-1 DNA? Are these fragments also broken into ~150 bp pieces, and in reproducible sequence boundaries?
Stevenson Ranch, CA

V. Randy White: The HIV pro-viral DNA is broken into 150 bp fragments. The key to Tr-DNA analysis is use of short amplicons, that way you eliminate the effect of minor variances in the sequence boundries. If your detection sequence is in the middle of a 150 bp fragment and you use a short amplicon, the fragment boundries can shift by 60 bp eather way without causing a problem.

Am I correct in assuming you would need to know the primary site to determine progression or response to chemotherapeutic/rad tx? What if primary is unknown? Any specifics to primary tumor?
Schaumburg, IL

V. Randy White: You are correct, you need to know the primary site. We are assuming here that in the case of prostate, that the tumor was removed. With the tissue in hand, you can genotype the tissue to determine its molecular signature. Potentially (not proven yet) you couly then monitor a patient's urine for reappearance of that molecular signature. This may eventually prove usefull for differentiating primary from secondary. For example, in prostate cancer patients where the prostate was surgically removed, it is not uncommon to find the patient 2-3 years down the road with a lesion in the lung - - is that primary or secondary to prostate? Tr-DNA might hold the answer if the molecular signature is the same. Hope this answers your question.

For the detection of CRC, slide 26 states that Tr-DNA provides the earliest detection of (Vogelstein-Fearon) progression. How do you see this fitting into the current health care system? That is, early tumor detection is required somehow followed by monitoring of Tr-DNA for evidence of APC fragments, etc? Will this rely on a better early detection method than currently available?
Stevenson Ranch, CA

V. Randy White: Great question. In that study of 200 people older than 50 years receiving routine colonoscopy, we did detect ACF that was missed on colonoscopy. Those patients tested positive for k-ras and p-16 methylation and on that basis those patients were re-examined with special staining techniques and the bright-stained colon tissue biopsied and confirmed as ACF. That would lead on to think that Tr-DNA might be used as an early detection method for colon cancer. But we are not prepared to make that statement. Significant additional clinical studies need to be conducted. In an earlier question I mentioned that k-ras is positive in several kinds of cancer. So I believe at this point in time that Tr-DNA might have a place in the arsenal of early detection tests, but not singly as a routine screening test.

It seems too easy: -sample: freshly voided urine. -centrifuge. -concentrate 100-1000x the supernatant urine. -run this specimen with your primers (amplify/detect). If it works... Is there any pattern in Tr-DNA fragments or they are randomly generated?.
Spain

V. Randy White: It does seem easy - - and it is. That is why we are so excited about this technology. There are differences in the Tr-DNA fragment margins and in an earlier question I pointed out that the use of short amplicons is the key. If you are looking for a marker in the middle of a 150bp fragment and you are using amplicons of say 25bp, then your sequence margins can shift by 50bp without generating a problem.

Do you think you'll be able to use TrDNA markers early detection/screening for a variety of solid tumors as suggested in Slide 26 on early stage Colon Cancer detection? That specific example looks promising.
San Diego, CA

V. Randy White: I think eventually we will be able to use Tr-DNA for early cancer "detection." However, at the moment we are only suggesting that it has a role in monitoring for progression and/or re-growth. The reason for this is that many of these markers are positive in other types of cancer. Until science discovers a unique marker for a particular cancer, or more likely a unique set of markers for a particular cancer, we are probably not going to be able to create a screen for colorectal cancer. However, there may be an opportunity to couple with one or more current tests to increase the diagnostic resolution of those tests. For example with the Exact Science fecal test for colorectal cancer, could the sensitivity be increased by simultaneously performing a Tr-DNA test for k-ras and p-16 methylation? We don't know, but that is probably where any near-term "screening" applications lie.