 |
 |
 |
|
 |
|
FDA Home Page | CBER A-Z Index | CBER Search | Contact CBER | CBER Home Page  |
||||
Simian Virus 40 (SV40:)
|
|
UNITED STATES OF AMERICA CBER-NCI-NICHD-NIP-NVPO SIMIAN VIRUS 40 (SV40): MONDAY, 27 JANUARY, 1997 Afternoon Session
The Workshop took place in the Natcher Auditorium, National Institutes of Health, Bethesda, Maryland, at 8:30 a.m., Kathryn C. Zoon, Director, CBER, presiding. PRESENT: KATHRYN C. ZOON, M.D. DIRECTOR, CBER ALSO PRESENT: DR. GALATEAU-SALLE
Introduction and Welcome by Dr. Zoon SESSION 1 Presentations:
Dr. Fanning SESSION 2 Presentations
Dr. Dorries LUNCHEON RECESS SESSION 3 Presentations
Dr. Hilleman
PROCEEDINGS
AFTERNOON SESSION 1:55 p.m. MODERATOR FRIED: We have a couple of more presentations of people who will continue from Session 1 this morning, who have positive and negative data on different detection of sequences in different types of tumors. In addition, we want other audience participation because you didn't really get a chance to ask any of the speakers questions this morning. So that we will go through a discussion and then you can ask the speakers and the panel will discuss various points on detection of sequences. So to start off, we'll have Robin Weiss give his presentation. Robin's from the Chester Beady Institute in London. DR. WEISS: Well, thank you, Mike. Mike Fried's asked me to speak first because I'm using the overhead and then we can rid of that as well as me. I'll just put up the overheads because we got involved, like so many others, into just looking to see whether there were SV40-like sequences in human tumors. Worked on by a student in our lab, Dave Griffiths -- it's all his work -- because we had DNA samples already and that the Institute and Cancer Research were adjacent to the Royal Marsden Hospital, which is a cancer hospital, and the Royal Brompton Hospital is the chest hospital. So there's quite an archival store of mesotheliomas. So that's what we starting looking at. And we've done nothing original. We adopted, in the first place, the primers within the T-antigen region that were described in the paper, New England Journal, by John Bergsagel and others that we've already heard about from Bob Garcea this morning, and one that is supposed to work for SV40 as well as BKV and JCV. And like Keerti Shah showed just before the coffee break, David Griffiths thought he'd better calibrate the primers first, and there came the first surprise. That if you take some plasmid or spike it with DNA, we find very different sensitivities. The primer 2 and the generic primer, they're primer pairs that are very sensitive -- we can detect between one and ten molecules of DNA -- but if we go to the shortest fragment, the 105 base pair fragment that is supposed to be specific for SV40, it's at least 100 times less sensitive in amplification. Paradoxically, the primer pair that's least sensitive gave us 100 percent positivity with mesothelioma, but we had to run to 40-plus cycles. So I think perhaps we need more discussion about the efficiency of the primers, and Keerti's talk was the only one that I really heard this morning that titrated that out against cos cells, which Dave Griffiths in our lab has also done. We also looked at semen samples -- this is whole, unseparated semen -- against samples we had prepared for a study of HHV8 in AIDS. These are all HIV-positive patients. And contrary to the situation from Ferrara, at least the Po Valley, we got zero. And these tissues were from three or four patients who died from non-malignant causes where we happened to pick up one sample. If we look at the mesotheliomas, these are mesotheliomas from patients who presented in London, a rather slightly different set from those in South Wales, then with this least sensitive primer pair we're getting some positive signal, if you go on cycling enough by PCR. And curiously, with the generic sequences, it ought to pick up all primate papomaviruses, polyomaviruses, we get many fewer. And with the SV40-specific sequence here we're getting only four. And so you use different sets of primers, you get different results. You go on far beyond the number of cycles you would need if this genome was in every tumor cell, and then you begin to get positive results. Our conclusions would say it's not clonal, we've not done immunochemical staining yet, but I'd be very surprised if they came out like Michele Carbone's, because there's simply not enough DNA there to get T-antigen expression in every tumor cell. But that's still to be done; we'll have to cut more sections. And we checked on the sequence for the four that were clearly positive, with the second set of generic primers. This is 105 base pairs and here's a prototype SV40. I think we amplified this out of cos cells. Here's the four mesotheliomas we tested; here's BKV, JCV. And whatever we have amplified is clearly SV40 over this small region, and in fact, there's only one nucleotide that's different from the prototype and they're missing this 9 base pair region that's in the two well-known human viruses. So there we are; that's our little bit of extra information which we can add to this analysis. I don't think it clarifies the subject at all; I think it further confuses it. But that's my feeling at this stage of the meeting, is that we don't know too much about what's there and there's a lot of variation between different labs. And I think the sooner we start exchanging blinded sets of samples so that the different labs can look at the same set and one central lab should then decode it, the more we might get to grips with whether these are technical difficulties, whether our positivities are false positives or whether there's a very low grade real presence there, and whether there are genuine geographic differences, or differences in collections. Thank you. MODERATOR FRIED: Thank you, Robin. So basically, when you do about 30 cycles you don't see it, and when you keep going you find it, is that the take home message? Have we lost Ellen Fanning from the panel? Okay, we also have some comments from Ethel de Villers from Heidelberg, who's been doing something and she will just tell us about it. She has no overheads. DR. de VILLERS: Thank you, Mike. Well actually, we came in from the cold because we've been working on papilloma viruses for many years now, and my main aim was to characterize and identify new papilloma viruses, and then we decided to broaden this to polyomaviruses as well. So we actually started off applying the methodology in a broad sense, to the polyomaviruses that we've been doing with the papilloma viruses. During the last three years we've been able to identify and partially characterize, 43 new papilloma virus types. So we were very optimistic about the polyomavirus types, but to tell you the truth, we haven't found anything. And I just want to give you a few details. I haven't got any overheads or anything; it was a quick decision to be here. But I think the experimental part is a very important one, and I think we heard very little about that this morning, and hopefully we'll have more discussion this afternoon. First of all, we started off using the VP conserved, amino acid conserved region, and we chose four different primers which we split up in degenerative primer pairs in order to identify all known polyomavirus types, including the mouse types: the kilham, the hamster, bovine, as well as the parakeet. By doing so we do 12 different primer combinations on one biopsy and we actually -- well, we think there should be more than two human polyomavirus types. I think many people have the same idea. We then looked at many different types of tumors -- the numbers are still small -- but I'll just mention what they were. We looked at normal lymphocytes of 12 samples, glioblastomas, five astrocytomas, ten cell lines of astrocytomas, five meningiomas, six lymphomas -- Hodgkin lymphomas, actually -- and ten lymphoma cell lines. Then we didn't only stick to the VP1 area; we constructed primers in the T-antigen region too, in the conserved region. We didn't find anything with that, either. At a later stage we included the Kaposi sarcomas -- we looked at 14 of them -- we did 20 bladder carcinomas. And by not getting any positive results we decided what we'll do is maybe go into the literature and try some of the primers that have been published. With great difficulties, with some groups we got hold of primers -- which was not the ones that they described in the papers -- but nevertheless, they gave us some primers and in the end I think the majority of people used the Bergsagel primers. We applied these primers in exactly the same way as been published, and I do not think one can consider the conditions of the PCR as stringent conditions. In other words, you do pick up other sequences, we do get a smear of cellular sequences in the background using those same conditions. If you use TEC gold you get a lot of bands, not only the smear. If we hybridize we get the smear hybridizing as well under those conditions. In some instances we got a little bit of a stronger band in the area where you would expect -- on this size that you would expect. What we usually do in the papilloma viruses is we clone and sequence. We are absolutely convinced there is no way you can get around that in any positive signal. So we cut out that area and we clone it and we sequence at least ten clones. We haven't found any polyoma viruses in any of these tumors or cell lines that we've looked at. What we did find is that we found, for example, many cellular sequences. One cellular sequence, for example, had 78 percent homologies to the Rb gene. We had another clone which had more than 70 percent homology to sequence in the fetal brain. So if you look in the databank you can find many sequences in varying homogies to these cellular clones. So that's the situation we are at now. We're progressing in this. What I would just like to mention is that what I miss in the data presented and as well as published, is the sensitivity which Robin talked about now, and on the other hand, our experience is that if you do not test your sensitivity by mixing your positive control to, say placenta background, then you get a different degree of amplification than you would use only the plasmid. On the other hand, all the negative controls for example, placenta DNA, water, and so on, are very often missing. The other thing is, we find that if we use more than 50 to 100 nanograms of cellular DNA input, we get a reduction in the efficiency of the PCR reaction. So those are just things that I would like to mention. MODERATOR FRIED: Thank you. I'm sure we'll cover some of these points more in the general discussion. Another presentation we have is by Harvey Pass from Wayne State. DR. PASS: Thank you, Dr. Fried. As you know, Michele Carbone is my collaborator and he starting working in my lab, which was SV40 negative before I left the NCI. And when Michele left to go to Chicago I was excited but also skeptical about these findings. In my unique position as a surgeon who takes care of patients with mesothelioma, I was able to recruit the first 48 for the first set of patients, but felt it would be necessary to re-establish this in a completely separate series of patients that were operated on by me at the NCI. That was done after Michele left and that would be done by people in my laboratory who essentially were learning the techniques. Could I have the first slide please? And I'd like the lights down please. Maybe I don't do the ethidium bromides as well as everybody. But we therefore took a series of 42 patients that were operated on since the first set, and not only looked at the amino terminus region, the Rb binding pocket for T-antigen as Michele has done, but concentrated on the larger fragment -- the 500 or so base pair fragment. But also with the help of Janet Butel, looked at the enhancer/promoter region for T-antigen and then also used primers to amplify the carboxy terminus in these patients, essentially. So we're concentrating on this primer here, primer pair, which amplifies a 574 base pair region of the Rb binding pocket which Michele touched on. Primers 7 and 8 -- that's my connotation of primers from the literatures that were described to amplify a 281 base pair of the carboxy terminus. And then finally from Janet's work, we use her RA1, RA2 to amplify 310 base pair region that was the regulatory region. Essentially, this just shows the primers. Essentially, this is the 7/8 primer which is the carboxy terminus, and then the RA1, RA2 here, so there's just the preliminary data. And again to refresh your memory about SV42, it essentially amplifies a larger fragment of the Rb binding region that when you look at your southern hybridization you may get two bands, one of which will reflect the presence of the centron and the other reflects that it is not there -- about 300 base pairs. Well, when we then did the ethidium bromides -- these are the positive controls which is a hamster mesothelioma tumor -- we weren't very impressed with SV42 on the ethidium bromides, but using the SV probe -- next slide -- here is the original. In the 42 or so new specimens you can see that we have some positivity, and in fact, in its 13 out of 42, which is 25 percent had -- we were able to amplify this region. And in some patients both species are present, but in most it's a single species. In the carboxy terminus region for amplification we found that 38 percent were essentially amplified using that primer, but again, we didn't have a probe for this so using BsaB1 digestion we took our positives, and this is the positive control, the hamster tumor that shows that it cuts, and then this is a positive that cuts, and this unfortunately is light, but another one that cuts. So it seemed like we had the same sort of amplification that we did in the control. So to reiterate, again, we found 38 percent positivity but again, with restriction enzyme digested, reflected what the controls were. No, we have not sequenced that. With regard to the regulatory region, using the primers described by Janet, we found very close to her data, about 50 percent seemed to be positive. And in fact, we had a unique restriction site here which we used Fok1 -- next slide. The positive control is here with an uncut, cut, uncut, cut, uncut, cut. Very similar in all these patients to the positive control, but we did sequence four of these patients that were positive, cloned out the product. Next slide. These four patients -- here is the original sequencing gel. To sum this up it was exactly homologous to what we found with H9A. But that wasn't enough. We wanted to go back and take another vial of tumor and then re-extract the DNA from another vial of tumor from these patients, and then do the digestion again to see if it corroborated our previous work. And indeed, when we re-amplified and then extracted a new specimen from those patients that were positive -- here's the positive control: cut, uncut, cut, uncut -- we found the same sort of digestion pattern. If you summarize all the data, then with these three areas this reflects the ethidium bromide data for the smaller fragment, 24 percent of the patients -- at least in the new series, the 42 patients apart from the original series -- have amplification of these three regions. I absolutely agree with the comments that have been made by the previous two speakers. I absolutely agree with the exchanging of specimens and standardization of this. Because the data that I'll talk about tomorrow which has to do with therapy, is going to be useless unless we find that this actually a true phenomenon. And I thank you for this time. MODERATOR FRIED: Thank you, and we have one more relevant to the last talk, by Dr. Galateau-Salle from France who will use one of the microphones. DR. GALATEAU-SALLE: Sorry for my transparencies and thank you to let me just give our result. We have looked for SV40-like DNA sequences in pleural mesothelioma, bronchia pulmonary carcinoma, and non-malignant pulmonary diseases that the study has been performed in Caen, France. We have studied 147 frozen sections including 15 mesotheliomas, 63 bronchia-pulmonary carcinoma, eight other tumors, and among them, one parietal osteosarcoma and metastasis, 71 non-malignant samples, and six mesothelioma cell lines. The DNA extraction was from fresh frozen biopsy and they were cut on ice under sterile condition, then was extracted by phenol chloroform method. Then amplification was performed with the primer designed by Bergsagel, amplified the conserved sequenced of large type and polyomaviruses, SV40 173 base pair, JC virus 129 base pair, and BK virus, 182 base pair. And to avoid false positive we considered OD index separated to 1.5. All samples were tested twice or three times. So we find positivity in 30 percent of bronchial carcinoma, 50 percent of mesothelioma, and 60 percent of non-menign pulmonary disease, and we find also the parietal osteosarcoma was positive. The DNA sequences were not related to BK virus sequences but three of our samples were also positive for JC virus sequences. The mean age of patient was 63 years old: the youngest was 41 and the oldest was 74. And the male/female ratio, we find 35 positive male patients out of 105, and six females out of 20. And if we consider persons of our sample exhibiting DNA-like sequences, a value of index according to disease, we find that in our adenocarcinoma, the OD index was higher than in mesothelioma, and we find that's all the peripheral adenocarcinoma, papillary carcinoma, or mesothelioma -- just adenocarcinoma was positive, and it was the same in non-malignant pulmonary disease. We find positivity in the peripheral line. And if we compare that mesothelioma to organizing priorities, we don't find any difference between the positivity in mesothelioma and organizing priorities. Now we have also studied the relation between asbestos exposure and SV40 DNA-like second positivities. We have studied on all the higher mesothelioma except one, where exposed to asbestos. And only 40 bronchia pulmonary carcinoma were exposed to asbestos and we haven't found any correlation between positivity and asbestos exposures. Now, regarding vaccination, it was very difficult because all the people have remembrance of the way that have been vaccinated and what type of vaccine. But all the people who were positive were old enough to have been vaccinated and born before 1963, and we haven't found any positivity in people born after 1963. The last result is, we looked for SV40 TAC expression by immunohistopathology and we haven't found any nuclear staining. Thank you. MODERATOR FRIED: And finally, before we start the panel we have -- Keerti Shah from Johns Hopkins will talk about BK in some brain tumors. DR. SHAH: May I have the first slide please? In this study we had looked for BKV-specific sequences in brain tumors. There have been a number of reports, most clearly from the group of Dr. Barbanti-Brodani from Ferrara, Italy, that they found BKV-specific sequences in human brain tumors, especially in glioblastomas. So we had done this study a couple of years ago and it has been published in Journal of Neural Oncology. We looked at malignant gliomas in 31 instances. We had purified DNA from frozen tumors, and these were obtained from Dr. Bert Vogelstein's lab. He had already processed them and we got the purified DNA. And we also got 47 paraffin sections from Johns Hopkins Hospital, and they were largely glioblastoma multiforming, but most all of them were malignant gliomas. We looked at them with two primer sets. This PEP-1 and PEP-2 are the ones which were developed in our lab by reactor, and those have been published. And then amplify 173, 176 base pair regions of T-antigen, and there are identical sequences here for both BKV and JCV. So we would amplify with a single primer pad, this one, and then hybridize with different probes; one for BKV and one for JCV. And this is the other primer, which is for the regulatory region of BKV which was used by the Italian group to detect these BKV sequences. So we obtained those primers from them. And these are the results. We were able to, by globin amplification, all of the 31 purified DNAs gave very good globin bands, and 44 of the 47 paraffin sections gave good globin bands. The sensitivity we thought was 100 to 100,000 copies of BKV or JCV, we would have picked up 100,000 copies total. And all tumors specimens were negative for both BKV and JCV DNA. From the tumors we had gotten from Dr. Volgelstein from which we had purified DNA, we could estimate the cell equivalent of tumor DNA, and we thought that we had at least 40,000 cell equivalents of the human DNA. And we thought that we would have picked up the viral DNA if only one of 40 of the tumor cells had a single copy of the viral DNA. And so the sensitivity was quite good. We still failed to detect the viral DNA in the human tumors. Thank you. MODERATOR FRIED: Okay. I would like to stop these more formal part of the discussion and the way I'd like to do it is shown on the first slide that I have. So we should be talking about: PCR conditions -- the sensitivity, the specificity -- since we have positive and negative; the methods of identification of the PCR products; the possibility of contamination where the people have SV40 or SV40 constructs in their labs, and what it means, the detection in normal and neoplastic tissues; what are the differences; why are we seeing this; and if there's any recommendations for the future. So before we go through and discuss with the different panel members the differences that they find about -- and the different PCR conditions and whether we would hope to, out of this meeting, get some sort of standardization -- John Lednicky will be giving a presentation from the panel about different technical details. John? DR. LEDNICKY: Well, as we all know, those of us who are looking for SV40, the samples are using PCR -- which is a powerful technique -- but our findings are often discordant. And knowing that many factors affect PCR reactions, it's likely that our results are affected, not only by sample choice, but also by specimen quality and PCR methodology. So I'd like to identify some problem areas with a goal of having this audience suggest ways to improve these tests. So I guess the central question to be addressed is: What is it about the PCR methodology that could be affecting the reproducibility of results among different labs? If I can have the first two slides, please. So particular questions to consider are shown in slide 1, and the first question I'm going to pose is: What's the most effective method to extract DNA from paraffin-embedded tissues? And we should consider: what PCR conditions should be used; are the primers really SV40-specific; how should we go about using and setting up positive and negative controls; and how should PCR products be verified? Now, a lot of people think they're PCR experts and they really don't have a feeling for what samples extracted from paraffin slides often look like. So in the upper panel here I've shown total tumor DNA extracted using -- chopping up tissue, doing a proteinase K digest, and precipitating all the DNA out looks like. And what you see in reality, although it hasn't stained, a lot of the high molecular weight DNA hasn't gone into this one percent agarose gel, and you see prominent mitochondrial bands here. In contrast -- and this comes as a very big surprise to people who have never looked at these, and the majority of labs never do this stuff, they just set up PCR conditions, assuming that they've recovered a certain amount of DNA -- and that is, oftentimes with DNA extracted from paraffin slides, you get very fragmented and degraded DNA. The reason we should of course, decide what might be the best way to extract these DNAs is, when you have fragmented and degraded DNA, this is really going to affect the PCR sensitivity; the efficiency of the PCR reactions are diminished. In these next two slides I'm showing some very basis problems. Now, we also need to be aware of problems arising from DNA preparation methods, and in this slide I show some purified DNAs that we received from other labs. In fact, this particular sample was hand-delivered to me by the person who supervises the PCR work in that lab. By the way, none of these DNAs were from Bob Garcea's lab; I just wanted to make that clear. So seeing such sloppily prepared DNA, how can you assume the DNA is not contaminated with other DNAs? And for people who are setting up positive controls based on amplification of alpha and beta globulin genes, how do you know what you're amplifying from samples like this, don't derive from the skin flakes of the technician working up these samples? It's a basic question, but it's something we really need to think more about. Now, with samples like this we really need to consider whether reliability is a problem if we subcontract PCR work. Like, what assurance is needed that the DNA is being handled properly? And here in the U.S. this is a very contemporary concern these days because, as my colleagues say, there are a lot of rent-a-techs in core facilities and maybe some sort of oversight is needed. Another type of DNA preparation problem is shown in this slide. And in this demonstration slide what I'm showing is DNA that was spooled from SV40-infected tissue. And what I've done is amplify the regulatory region: these two are control lanes; this is a negative control lane; this is a lane that has total DNA, just precipitated from a sample. Here I've resuspended the spool DNA and as you see, I don't get any signal, whereas what's left in the tube does give me a signal. Now, spooling is still used by many labs working with eukaryotic DNA and I'd like to note also that working with coded samples and not knowing beforehand how a sample was prepared, in our lab we have not just detected SV40, JC virus, or BK in a single spool DNA sample we've looked at. So I'd like to discuss PCR conditions and to demystify some of the methods our own lab has developed. Now, a primary question when PCR signals are seen is: Is it really SV40? Now, our lab's approach is first of all, look at more than one site of the SV40 genome. So we look at some of the sites. We typically look at the regulatory region, Rb proximal binding site, carboxy terminus of T-antigen. And in particular, the carboxy terminus of T-antigen shows variability between SV40 strains and sequence data from the site may be useful for taxonomic and epidemiological studies. Now, other sites such as the regulatory region, are useful when the target DNA is episomal, and that the regulatory regions of SV40, JC, and BK are distinct. In this slide which is pretty busy, I'm just showing primers and PCR, annealing temperatures we use. Now, one of the commonest questions we're asked is, why do you use so many cycles? And when you're working with paraffin samples, why do you use so many cycles? Well, when you have a lot of fragmented DNA, I guarantee you, you need to use more than the standard 30 cycles that a lot of people normally use. And here what I've listed is two temperatures. The temperature in parenthesis which is lower than the one to its immediate left is the temperature we use when we're working with samples extracted from paraffin. So notice, these are what I refer to as lower stringency conditions. We have found that it's not possible to use more stringent conditions, and a lot of labs seem to do this. Now, very importantly, since more than 40 cycles are needed, we should discuss detection sensitivity as some people have said earlier, because a lot of labs overestimate their detection sensitivity. And the biggest problem is they use plasmids without spiking them with additional DNA, and that really decreases the sensitivity. Can I go back to the other slides? Now, I'd like to give a warning -- and it's not a good idea at all to use lower stringency conditions when you have highly intact DNA -- and this is something else a lot of labs do. And the reason for that is numerous, non-SV40 PCR products are formed. And we have also actually sequenced some of those bands and confirmed they're not papomavirus bands. So the problem is setting the appropriate PCR conditions for these samples derived from paraffin samples is really more of an art than a science now, and we really need to put our heads together to try to come up with, you know, realistic protocols. Additionally, the conditions cannot be universally applied, and in particular -- for example, when we use these two primers we have to use a lower, what I call high stringency condition. And the reason is, with these particular primers which amplify the carboxy terminus of T-antigen, if we go much higher than 60 degrees, we get truncated T-antigen products in addition to the full length product. So you have to be careful about some of these conditions. The next two slides, please. Now, another reason for using different sets of primers is that it's possible that DNA sequence changes might occur in different strains of viruses, and in particular, the regulatory region of these viruses might be somewhat different. The primers we use seem to work for different strains of SV40 even those with rearrangements like SUPML-1, but there is a danger, and I'd like to bring to everyone's attention that the primers being used might not be specific for SV40, even though very sophisticated computer programs tell you that they would, under the conditions you want to use them. And so for any set of primers you have, you really have to test them. It's very hard to use computer programs to really predict whether they're going to work. So for example here, using RA3 and RA4 primers which have quite a few mismatches with JC virus, even under relatively stringent conditions, we're actually able to amplify the JC regulatory region. Here I've amplified the med1 regulatory region and sequenced it in both directions. So we find that we can actually amplify JC and BK virus. The point is, these findings highlight the need to verify the identity of PCR products. You can't go just by seeing a band on the gel. Another important question is, how do you distinguish between true positives and false positives? Now false positives can usually be traced to contamination by controlled DNAs, and our lab solution is to substitute SV40 templates for natural templates. And in this slide, what I've done is create some SV40 templates which have unique XHO, or SAL 1 sites. So when you amplify them, the product is about the size of what you'd get off a natural template, but then now you can do an XHO 1 digest and only the artificial template gets caught by XHO 1. And we're developing similar constructs for other regions that we analyze and we think this is a really good idea for people to use for positive controls. Now, another approach we're trying to perfect is that of using long PCR to amplify the whole SV40 genome. And this slide shows some of our findings. Using a commercial kit we're able to now amplify an entire SV40 genome from plasmids environ cell lysates. And the procedure, the way we use it, works fast. If you remove some of the high molecular rate DNA first and then do your amplification -- I won't go into any more details -- but I think this approach eventually may be useful in that it will be possible to not only answer whether episomal DNA is present, but also because it will be possible to amplify the entire genome for cloning and additional analysis. So I'd like to discuss the merits of DNA sequencing. So in this slide the sequence DNA band -- I'm sorry, the sequence PCR DNA band is clearly different from that of the control template. There are two changes done here, but if you scan up here you'll see that there are indeed, changes. Now, the question is, how do we know these changes aren't artificial? And if you look at this slide, the answer is evident in that you see repeating patterns of 9 base pair deletions or insertions that aren't seen in our template for control positive DNA. Also, the sequences we've come up with are different from the standard SV40 strain that's present in our laboratory, which is the Baylor strain of SV40. We have found that just merely doing southern blots may be a tricky thing, because as one of the speakers said earlier, if you play around with the hybridization conditions, you actually non-specifically light up unrelated DNA. And I hope this presentation put some of these problems in perspective, and thank you for your attention. MODERATOR FRIED: Thank you, John. You've opened up a lot of different aspects of the PCR technique that I think we should discuss. Could I have my next slide? So what we have discussed, we have some positive results, we have some negative results, but what's quite clear is that if there was one copy of SV40 per cell, per tumor cell or per normal cell, it would be very easy to detect. And it's not very easy to detect, so there's probably a very low level or the primers people are using are not very specific. So the question is, what are the limitations, how can we increase the sensitivity of the PCR and the relevance of the copy number? Could I ask the different members of the panel how much they think they're detecting in copies per cell? Does anybody want to volunteer? Michele? DR. CARBONE: We have done the original experiment that's indicated. We are able to detect one genome in our PCR reactions. I would like to comment just briefly on what you just said -- MODERATOR FRIED: Sorry -- one genome per what? DR. CARBONE: One SV40 genome. MODERATOR FRIED: If you have in your whole PCR reaction, one genome, you could -- DR. CARBONE: One SV40 we've detected. Actually, ten genomes. At one genome -- between one and ten genomes we are able to detect. Now, the real problem -- not the real problem, but one problem that should be considered in what you said about one copy of the cell is that here we're not talking about cell cultures, we're talking about tumors. And obviously, in a tumor we have -- the majority of the cells often are not cells that are tumor cells. Many of them are reactive cells that are not malignant cells and other cells are necrotic cells. So that should be considered when we talk about the level of sensitivity. MODERATOR FRIED: Fine. I would agree with that, but still, I mean, I'm sure we're going to get to the cancer whether this is related to cancer in other sessions, but it's clear that from all the other papomavirus or the polyomaviruses, we know they don't get lost -- I mean, they stay there. And the only reason that possibly an episomal copy would be doing something if it got into a cell and excited that cell, stimulated that cell to secrete factors which would make other cells divide, so then you could have low copy number. But certainly, you know, if it was cancer cells we would have plenty of copies, I think. DR. CARBONE: We have at least, however, one bovine papilloma virus, type was 4, that get lost. MODERATOR FRIED: So Dr. Shah, how many copies do you think you could detect, or not detect? DR. SHAH: I think we detect perhaps, ten to 100 copies, genome copies, of the virus in our PCR reaction. Not per cell, but of the virus. Just as -- MODERATOR FRIED: The whole PCR realm -- DR. SHAH: -- Michele say is one to ten, I would say ten to 100. We believe that even with paraffin sections we are processing at least 1,000 or 5,000 cells. So we would detect, if there was this one copy in ten cells or 50 cells -- if there was one copy of the viral genome in ten cells, I think we would detect it. This would take care of the problem that Michele described, that not everything in the paraffin section is tumor cells. MODERATOR FRIED: But sometime you have fresh tissue also. I mean, have you not -- DR. SHAH: We had fresh tissues -- we did not have fresh tissues for the mesotheliomas, but we had fresh tissues for the brain tumors. For that we were -- because we knew how much DNA we were processing, we thought that that was DNA which would come out of something like 40,000 cells, but that was with the BKV system. So if we can detect 100 copies and we are 40,000 cells, then we can detect -- one complete genome in 40 cells was our estimate for the BKV study. MODERATOR FRIED: But you're not detected what other people are. I mean -- DR. SHAH: That is true. MODERATOR FRIED: And you're also not using radioactivity hybridization, you're using biotin. Do you think that's less sensitive? DR. SHAH: I don't think so. We used to do radioactive probes until about three or four years ago. When we changed to the biotin label probes we examined it very thoroughly and also solves the experience of many people in the papilloma virus field. We do this routinely, hundreds of times, for studies on human papilloma virus in cervical cancer, and there is no loss of sensitivity by moving to the non-radioactive detection system, that we have observed. MODERATOR FRIED: John Lednicky suggests that most of the DNA they're detecting is episomal -- I mean, not integrated. I mean, is there anything that possibly -- DR. SHAH: No, we do not precipitate the DNA. We proteinase, extract it, and test it in the same tube. So we do not have this problem of spoiling and losing some portion of the DNA. MODERATOR FRIED: Could anybody else suggest why they think there's differences between positive and negative? DR. TOGNON: Yes, I would like to comment on the sensitivity. Just to have a rough idea how many molecules in terms of genomes we have in our detection experiments, we did a sort of reconstruction experiment. Since we start always with 500 nanograms of human DNA we mixed in dilutions different amounts of SV40 DNA. And at the end, it turns out that we have the sensitivity of around ten molecules in our assay. I would like to say something else about the negative results that we heard before. I wish to point out once more, the problem that's related to the extraction of DNA is not enough to digest with proteinase K and SDS. We usually extract several times with phenol and chloroform the DNA, and at the end instead to precipitate the DNA, or instead to directly amplify the DNA, we dialyze for two or three days, the DNA. This is very, very important because usually the BK DNA or the SV40 DNA, or JC DNA usually in episomal state. And the amount of the DNA of the viral origin is always very, very low. We estimate in our assay is approximately .1 fentagram. That means practically nothing. MODERATOR FRIED: But it's been pointed out by Barielle and discussions, one of the classic ways people purify plasmids or SV40 or circular molecules is by precipitating the DNA, so why do you think you'd be losing the episomal, especially when you have so much carrier DNA to bring it down? DR. TOGNON: The difference is the amount, because if you precipitate your DNA, you precipitate the high molecular weight DNA. If you have enough episomal DNA, episomal DNA can't precipitate with the human DNA, but if the amount of the episomal DNA is very reduced, you've lost the DNA and you've lost the signal during the PCR. This is a very, very simple experiment, because you may reconstruct in the laboratory, okay. You can make ten or 20 different Eppendorf tubes with different amounts of your episomal DNA together with the 500 nanograms of human DNA. And at the end you eventually can repeat the extraction and you see the difference. MODERATOR FRIED: Dr. Gibson? DR. GIBSON: This is something to consider, and we learned this by trying to purify DNA using a number of commercial kits. But a lot of people lose even mitochondrial DNA when they're precipitating or somehow collecting their high molecular weight DNA. So as a rule of thumb, I think it's a good idea to look for your -- to see if your methods are bringing down the mitochondrial DNA. So mitochondrial DNA is circular, it's about 16 kB, and any purification methods that will work for SV40 will also work for mitochondrial DNA. But we have actually gotten samples from other labs that have a lot of high molecular DNA and you just don't see any mitochondrial DNA. And I'm sure if the SV40 or whatever out there -- papomavirus you're looking for is in there in an episomal form, you'll never see it. MODERATOR FRIED: What about increasing the sensitivity? Ellen Fanning, did you have some points to make? DR. FANNING: Well, I was wondering, maybe some of the people who were doing this routinely could respond. Whether it's not possible to construct a competitor molecule that uses the same primers and thereby eliminate the variable activity of different primers -- the efficiency with which different primer pairs will amplify a target sequence. You could construct a competitor. There's some effort I guess, already in that direction, which has a different size or which has a restriction side or something like that, so that you could distinguish it from the products that you're trying to look for. MODERATOR FRIED: That that would help avoid contamination of people who are putting in SV40 to use as the primers. So you would put a primer, some junk DNA, whatever, and the other primer. And this could be any size. And you could spike this in to just check, you know, and it wouldn't be falling off people's hair because it could be something else. What about other -- you're also suggesting maybe, other types of PCRs in situ? DR. FANNING: One other thing that one wonders, particularly with tumor cells that appear to be staining for T-antigen, is whether those cells couldn't be used. Certainly, the tumor cells could be distinguished as tumor cells when you look at them. For example, do in situ PCR on tumor cells and ask whether those cells, rather than the contaminating, normal tissue around them, may be the cells that are specifically containing the viral sequences. Is this feasible? MODERATOR FRIED: Anybody? DR. CARBONE: I show tomorrow, some RNA hybridization. We did not do in situ PCR on the cells. We hope to do it soon, but there comes one problem, if I can address that. I mean, I agree 100 percent with John Lednicky, what he said. He presented an excellent presentation of what should be done, and I'm sure that if we do what he says it's always going to work. It fact, it works in our lab and in fact, that's the way that we work and that you have seen the presentation of Dr. Pass, that's the way he's working. But then comes in terms of practical problems and that is, that sure, you want to use many primers, you are using in situ hybridization, you want to use as much as you can. But all this takes time, it take money. And that has to be taken into consideration because it's a factor that has affected this research considerably. In other words, if you want to test 100 samples and you want to test 100 samples with primers for many different regions, you need to have the resources to do that, otherwise, it would be impossible. A more practical approach that also has been taken is the one of sequencing the DNA. I think, in my opinion, that that's probably the best approach if you don't want to do extensive studies, meaning using a lot of primers, a lot of hybridization, and a lot of work. Not because you don't want to do it, but because you don't have the resources to do it. And for example, if I can answer to what, the excellent presentation that Robin Weiss gave before in which he showed something that I think is in fact, the point of the discussion today. The point is, he said I use different primers for this before, and here I'm getting different positivities. I go from 100 percent when I use SV3 primers, to -- I don't remember how much -- when he used the beef primers, to something less when he used longer primers. That seems to be the argument, not that much why some lab is not finding it, because it seems to me that overwhelming we are finding it. But why is that using different primers we get different percentage, and what primers, what set of primers should be used? My experience has been that initially we used that set of primers that gave the 100 percent positivity that was reported before, or the other one that is called beef set of primers. These are shorter primers. The problem that you can have then, is that you have to rely to hybridization, and also the problem of the temperature was brought up. When you rely on hybridization there can be cross-reaction with BK or JC, and you hope that in fact, what you're seeing is true, but you cannot be absolutely certain. And that's why in our last paper, together with Bob Garcea, we went to the longer primers, that they are the SV two sets of primers -- because in our experience, at least using that set of primers, is big enough that you can be more assured that what you see in there in hybridization is in fact, true SV40 DNA and not something else. If in fact, you're using the shorter primers, the one that used beef primers, for example, in my experience you need to use those primers when you use for example, for my fixed tissue, because you can't amplify 574 base pair many times, so you need to go to a shorter primer. Well, in that case, rather than relying on an hybridization where you always can question, was 58 degrees enough, should we go to 60 degrees, you can just do direct PCR sequence. You have cloned your PCR products and checked that. So shortly, what I was suggesting that the approach that John suggested is the ideal approach if one has the resources to use that approach. If one does not have the resources to do that, the alternative is to use the set of primers that gave the lower number of positive results but still gave positive results that are the SV2, SV ref set of primers that we use for example, in the Oncogene paper. Or if you're dealing with the formalin fixed DNA, then use the beef set of primers that will probably take also BK or JC, but if you sequence it you should be able to distinguish among them. Then the question, why you get more positive when you use the beef set of primers or you use the longer set of primers? I don't know, but obvious we have an explanation that seems plausible and that is, that those primers may well cross-react with BK and JC. And so it is possible that when you're using that set of primers you have seen, not only SV40, but you are seeing BK or you are seeing JC. And the only way to know that would be to sequence the DNA. And I took too much time, I think. MODERATOR FRIED: People from the audience? Ethel? DR. de VILLERS: I would just like to make a comment again regarding the papilloma viruses, and I think we're not so far away -- MODERATOR FRIED: Is that microphone on? DR. de VILLERS: I hope so. Is it on yet? Now? Can you hear me? In the papilloma virus work we've done, we do not find any difference whether you spool the DNA or where you precipitate it. We do precipitate the 8 kilo base pair plasmid with the DNA, and we do spool it out if we spool out the DNA. I don't think there's that much difference between the five and the 8th Kb fragment, or the episome. And the other question is, or the other thing is that I wanted to mention, was that if you -- well actually, I want to be mean because what I wanted to do is make a comment, what I read last week in the "PCR Protocols", in the small book where they quoted Cary Mullis. And he said, if you need more than 40 cycles to amplify a single copy gene, then you have serious problems with your PCR. And I just want to mention that our PCR that we're using, we go down through one genome copy per cell in our detection method, and we still do not find any polyomavirus in these tumors. MODERATOR FRIED: Thank you. Is there anybody else from the audience who would like to make a comment? DR. VILLARREAL: I wanted to comment. I'm Luis Villarreal. I've been studying episomal states of polyoma, mass polyoma for about ten years now, looking at low-level episomal persistence, about one copy per cell. And this problem that you've encountered of physical conditions for the purification and precipitation of the DNA strikes me as odd. I've never seen that as a phenomenon. And I suspect the situation may not simply be the size of the DNA but the way it's being handled: the precipitation g forces involved, the salt conditions, etc. There are a lot of other variables that affect the yield. So that's one thing to consider. I guess I'll let the other speaker for now. MODERATOR FRIED: Do you want to go up to the microphone? Could you identify yourself? DR. OXMAN: Mike Oxman, San Diego, pre-historic SV40. I have two questions. One is, if you're talking about polyomavirus tumors, one wouldn't expect episomal DNA; one would also expect integrated DNA. So that may not be such a big problem. The other question is, I would love to hear the people who are using more than -- who are showing fluorescence or immunoperoxidase stains in which a number of cells shows T-antigen, and are still using more than 40 cycles. And I wonder what the explanation is for the need for that many cycles? MODERATOR FRIED: You'd like to answer? You had 50 percent? I mean, you showed one where there was a lot of T-antigen -- DR. TOGNON: Sure, sure. I'll answer, first of all, to the problem related to the papilloma virus. We have similar experience. We don't have any problem with the papilloma virus. Indeed, the number of genomes of the papilloma virus in the tumor samples is always higher compared to the -- in my experience, in my experience -- is always higher compared to the polyomavirus. And for the presence of integrated state of the different polyomavirus, we found that usually the percentage of integrated polyomavirus in the tumor DNA is always very low; let's say approximately 20 percent of all the sample. In that case of course, it doesn't make any difference because the DNA is integrated in the human genome. And for the presence of the antigen, the various breaks in the cell lines, the polyomavirus -- SV40, JC, and BK -- usually infect the cell in foci. If you have for example, 110 cell -- 106 cells, only let's say, 100 on 1,000 are infected and expressed the last T-antigen. So you have always the foci of discreet presence of the last T-antigen, but not all the cells express the last T-antigen. DR. BUTEL: Mike has asked a very important question relating to the integrated state of the DNA in these samples. And the fundamental answer is that we don't know whether it's integrated or not. We have not had enough sample size to do the right experiments to tell whether there's any DNA integrated. I mean, it's conceivable that we're detecting episomal DNA, it's conceivable there would also be integrated DNA, but we haven't been able to do those experiments to answer that question. DR. SHAH: I think our real problem is not so much to increase the sensitivity of the assay, because everyone seems to think that they're detecting one copy in ten cells, whatever. Our real problem is to make sure that our specificity is good. And I don't know of any DNA tumor virus where you could not detect the genome by non-amplification-based assays. What is desperately needed is to take some of these positive samples and see in a simple certain hybridization without amplification, whether you can detect the correct bands or not. I think that is really needed. DR. BUTEL: There's not enough DNA when you're just dealing with one tiny little amount. DR. SHAH: Yes, but -- DR. BUTEL: If we had larger pieces of DNA than those experiments -- DR. SHAH: How many micrograms of DNA is obtained from these tumors? DR. BUTEL: We've only been dealing with, you know, a paraffin slice. MODERATOR FRIED: But now you know what you're looking for. You don't have to go back to archival material. I mean, there should be more material that should come up right away. DR. CARBONE: May I intrude into this discussion? Could it be possible that now that we have a new technology -- that I agree with you, that before it was not possible to -- the DNA tumor virus was detected by southern blot, but that was also true because there was not PCR. Today we have a new technique, and so given the fact that we have a new technique that is much more specific, it is very possible that today we are able to detect things that in the past we simply were not able to detect. And actually, if you look at some old papers -- there is one in PNIS; I think it's Krieg, the first author; I'm not 100 percent sure -- he shows southern blot showing that brain tumors contain SV40. But the bins are dirty. I have done the same southern blots and I could show those southern blots and I believe that depending whether the reviewer is a friend or not, he could believe it or not. In other words, the signal is not that strong that you can sell it for sure that the signal is specific. But certainly, you'll see something there. And what I'm suggesting is that today we have a new technique -- the polymerase chain reaction was not available ten years ago -- and we may be seeing things that before was not possible to see. MODERATOR FRIED: Yes, I think PCR is obviously more sensitive than southern blotting; I think there's no doubt. And I mean, the question is, because there's positive and negative, can we get to some consensus where maybe an agency would send out different cells blind to the different labs and, you know, standard sets of conditions that people could look at them and come back, or people can contribute to -- DR. CARBONE: But this would meet -- Bob Garcea, Dr. Pass, and Dr. Procopio just did and published in Oncogene. MODERATOR FRIED: That's right. DR. SHAH: May I suggest? There's a strategy which has been proposed by Howard Strickler from the NCI, which I think really will address some of these problems, which will examine the different labs and the ability of the labs to reproduce their results. I think that would clarify much, and I wonder if Howard would comment on it? DR. STRICKLER: My suggestion was, in the face of the uncertainty of the data, that what we really need is an exquisitely controlled third-party study. The Oncogene study was a very nice project involving four different laboratories, but it's somewhat difficult to follow exactly where DNA was extracted, who handled the samples, which laboratories worked with them. It wasn't -- considering how important this issue is and how easy it should be to clarify these questions, it seems that really, we should just move forward and do a study in which multiple laboratories, using their own methods, test specimens, and we can directly measure the intra and interlaboratory reproducibility of the results, and we can talk about the results afterwards. As long as I'm up at the podium though, I'd like to address a question which is, in those laboratories in which positive findings are being obtained, doesn't the extreme sensitivity of your own assays concern you? There's only one study so far which presented data suggesting an approach where they examined whether or not the virus was actually in the tumor cells. And it's amazing to me -- unless I'm missing a point, which could be -- that in situ hybridization data isn't available yet, the PCRs are picking up what seems to be low copy numbers. Maybe SV40 is there. Is there additional data that someone can point to suggest that these viruses are actually in the tumor cells? MODERATOR FRIED: Michele? DR. CARBONE: I'll answer your question. I'll show some in situ hybridizations tomorrow. I wouldn't say that it's so amazing that no more data are being presented because again, I don't want to act like I am baby. But we have been working with no money, and when you work with no money you can't expect too much. And actually I think that working with a little amount of money that we're working, we have produced a lot of results. The other point is that here, everybody seems very concerned about these 40 cycles. Now, I have to admit my guilt here that I've never tried 20 cycles. And the first thing that I'm going to do when I go back to the lab is to check what's going to happen if I do 20 cycles or 30 cycles. I didn't think that this was such a big issue. The point was, if there is not there. Once something is -- if something is not there, I mean, if I take a negative sample I can amplify 100 times; still he would remain negative. So 40 cycles, I'm not sure that that's certainly the limit. And for the question that the Doctor asked before, saying, you see that in immunohistochemistry, why you need to do 40 cycles? Probably for those samples I don't need to do 40 cycles; it's just the standardized thing. You have a number of samples, many of them will not look that good. Of course, one shows slide that is the best slide is not going to come here and show that slide. So why show a sample that shows a lot of positive cells? And I'm sure -- not I'm sure -- I think it's a plausible question, it's possibility that if I go 20 cycles with that sample I'll get it. MODERATOR FRIED: Okay, why don't you do 20 cycles and come back? DR. CARBONE: I'll do. MODERATOR FRIED: They're lining up on the microphone there first. Go ahead. AUDIENCE PARTICIPANT: The primer pairs that have been used so far are very interesting and they are pairs for regions that are control regions of T-antigen and of the enhancer/promoter region, which interact with cellular components and are likely to have cellular analogs. It would be interesting and I think increase my confidence in the data, if you use sequences to viral structural proteins like VP1, which would not have cellular homologs and which would still be very good at detecting BK, JC, and SV40. DR. BUTEL: We did VP1. MODERATOR FRIED: Yes, there may not be some conserved, so you don't really know -- DR. GARCEA: I would love to find the cellular homolog to the Rb binding pocket of SV40. I'd switch my projects over. DR. LEDNICKY: I think he raises a very important point. And actually, we would also like to do more of those studies but there is a problem; there's only one strain of SV40 that's been fully sequenced, and we need to increase the database -- our lab's beginning to do this. We don't know, for example, that there aren't other serotypes of DNA, and for people who are looking at antibody reactions there might be something we're missing, for example. But that's a very intriguing point. MODERATOR FRIED: Hopefully, with John's long PCR, then you'll come around and go through both the control region and the viral protein, so satisfy everybody. Bob? AUDIENCE PARTICIPANT: Two trivial questions -- DR. GARCEA: One thing I want to point out about that. I mean, I found that one of the most striking results -- I mean, I'm a complete skeptic, and it's just surprises that make me less skeptical. But one of the biggest surprises was finding 172 base pair repeat. That is simply diagnostic of a virus that's come very soon out of an animal. I mean, Ron deRogers has shown that. So I think that that is a very -- MODERATOR FRIED: But on the other hand, Michele found two 72 base pair repeats. DR. BUTEL: I wanted to respond to Howard's comment though, that there is very little new information here. I would disagree with that. We took a different approach in our study instead of just continuing to look at more and more samples. And that was to try to look very carefully at the sequences that were being detected, because we too, were very concerned about whether there was some odd, contaminant that was being picked up that was slipping in from somewhere -- even though we're very careful to always do negative controls and we set up the experiments in different room and do all those kinds of things. But I think the bottom line is, when you sequence and you find one 72 base pair repeat, and we don't have anything in the lab like that, and the variability that has been discovered at the end of the T-antigen gene which doesn't correspond to any laboratory viruses or to the sequences that we had found in the brain tumors -- we found different sequences in the few osteosarcomas that we looked at. And so I think that is new information and it says that there are -- in my opinion it suggests that there are different strains out there that are somehow or another, present in the tumors that are being examined. MODERATOR FRIED: But we're limited by our primers of what we're going to detect. I mean, if we don't have the right primers we're not going to see -- DR. BUTEL: There are going to be other things that are not being detected -- DR. GARCEA: One more quick thing before -- I'm sorry -- because Janet failed to mention it in her talk. When we gave her these samples to transfect into cells, they were all blinded, and only sample number 12 gave a virus out. When we decoded those samples, samples one through 11 were from paraffin block specimens. Sample number 12 was the only fresh tumor specimen. I just want to point that out. MODERATOR FRIED: Okay. Bob? AUDIENCE PARTICIPANT: Two trivial questions. One is, why don't you throw in a set of primers totally unrelated -- say, hemoglobin primers -- and see whether or not in the same reaction -- in the same reaction, so you always have within the same reaction, you know your PCR reactions work. Number 40 doesn't bother me in the slightest. I've seen coli, repeatedly giving a negative result when the sequence is there. So that way you'd have an internal control. In every single one you get another -- you get a -- some other size. MODERATOR FRIED: That was suggested. I mean, the people -- AUDIENCE PARTICIPANT: Yes, just throw them in the same -- MODERATOR FRIED: But I mean, there's always a chance that it comes from the operator, if you're looking for human. I think maybe what Ellen was suggesting, putting primers on blind pieces of coli -- AUDIENCE PARTICIPANT: Yes, but then you get the same problem with the contamination from the blind primers. You can do it either way. That's fine; yes, I agree. The other thing is, in terms of knowing whether or not it's free DNA or not, why don't you use the complements of the same primers you've used and run them in the opposite direction? You'll get multiples of unit length SV40 if there's full length SV40, and you'll know. MODERATOR FRIED: That's what John was saying. DR. LEDNICKY: This is one reason we're trying long PCR. But keep in mind that when you're working with archival samples, there's a limit to the size of the DNA that you cam amplify, and standard textbooks will say, 500 base pairs. So we found this is true and in fact, our signals in general, decrease with respect to the size of the amplified product -- archival samples. AUDIENCE PARTICIPANT: I would like to make a question to Dr. Shah about the extraction of the samples from mesothelioma. When we made the first experiments with Michele that had been published in Oncogene, we were using only fresh tumors. Instead, when I went back in Italy and I start to look at the statistic that is in paraffin-embedded tissue, we had a lot of difficulties and it took a while to sort out why the first screen we had positivity and the second screen we had negativity of the same samples. And it came out that it was crucial for us, how long you take your sample after extraction. This maybe, was just a problem in our lab, maybe. The DNA was not completely destroyed so we were getting results after extraction but not later on. But I would ask you if you are sure that this could not affect your results? DR. SHAH: We tested the specimens soon, very soon after the proteinase K selection; within one or two days. AUDIENCE PARTICIPANT: Within one or two days? This is the problem. Within one -- two days we were not able to get the same quality of results. DR. SHAH: I think it is quite true that if we have fresh tumor DNA we would have a better chance of finding something which is already there. We have the controls for the globin amplification, and we used this thing very extensively in many other studies. So this is not the first time that we were doing this. AUDIENCE PARTICIPANT: Sure. However, I would like to point out that the control -- also we are running the same controls, but these controls are not the best we can get because you know, you are comparing genomic with maybe episomic material. MODERATOR FRIED: I don't know about these things, but do you need to use archival DNA? I mean, if you know what tumors you really want to look at, are they not able to that fresh anymore? DR. SHAH: I think it would be wonderful to take fresh tissues; there's no question. DR. GOEDERT: Jim Goedert from NCI. I'll comment on that. The tumors you're talking about are extraordinarily rare. I mean, they're going to be hard to find except at very major places where the lab is close to the clinic. I actually wanted to raise a question about specificity. I was very impressed by the sequence data that Dr. Butel had presented and others, and I think that's usually considered the gold standard. But I wanted to ask the panel members or others in the audience, whether they thought there was a possibility that artifacts, either in the amplification process or actually in the sequencing, could draw some question as to the specificity of those results? DR. BUTEL: Let me answer. One reason, I don't think that there's a big problem with PCR artifact. If you consider the brain tumor sample 12 where we had the information, say the T-antigen sequence based on what was in the tumor, and then after the virus was rescued and it was sequenced, the sequence for that part of the gene was exactly what had previously been determined in the tumor specimen. So certainly there is an example of where there's no PCR artifact involved. And it would seem that there would be artifacts popping up in the other gene regions as well, and there's not been any variation found in the fragment of VP1 that's been amplified, for example, or any changes in the Rb domain. MODERATOR FRIED: So there's no PCR sequence -- I mean, could you say all your sequence differences are due to -- DR. LEDNICKY: That's a question we get asked a lot. And this concern -- people shouldn't be overly concerned with this in that, yes it's true. If you clone the DNA that you PCR amplify and then sequence that, certainly you'll see spots where you'll have possibly artifactually-induced changes. But if you do a direct sequencing reaction on the primary PCR band and run that out in a gel, you'll pretty much be able to tell what the sequences -- you might see small bands showing up occasionally where probably there was exactly that -- the change at certain sites. DR. WEBER: Thomas Weber, Hamburg. Maybe I may lead you back to the question of quality control. Under the auspices of the European Union, we have done a quality control study on the amplification of JC virus from one biological fluid, which is CSF which may be different. These samples were sent out to nine laboratories throughout Europe and it didn't matter what kind of extraction method the laboratories used, it didn't matter what time it took from the central laboratory in England to come to the receiving laboratory, whether or not the colleagues detected the DNA or not. What came out basically is, like in your report, that using primers centering around the T region, you are about by a factor of ten to 100 more sensitive than taking from the late gene region. That was the down to earth message. So I can strongly encourage and urge you to develop a quality control panel for paraffin-embedded sections, and I think once you have established that, you should go out and do sequencing, not jump ahead or do sequencing first before you haven't done the quality control studies. MODERATOR FRIED: You said you send them out to nine different labs? DR. WEBER: Nine different laboratories -- MODERATOR FRIED: Was it consistent -- the results? DR. WEBER: The results were consistent except for one laboratory that detected JC viral DNA in every sample and the dilutions were like, from one million copies per hundred microliters, to .001 copies. And they detected it everywhere. So that one laboratory had a contamination problem. The other eight laboratories detected between one and hundred copies per hundred microliter of the sample, or ten microliters of their reaction. MODERATOR FRIED: Somebody in the back? DR. LOWE-FISHER: My name is Barbara Lowe- Fisher and I'm cofounder and president of the National Vaccine Information Center, which for the past 15 years has been representing consumers who are concerned about vaccine safety. Before I ask a question of Dr. Garcea I'd like to commend the organizers of this conference for bringing together independent researchers to talk about their meticulous research into the possible role of a monkey virus in human cancer. This is the kind of quality research that deserves recognition and priority funding because it could someday lead to more effective cancer therapies. I'd also like to say that parents across America are contacting our organization and they are not as concerned about whether or not you've proven, beyond a shadow of a doubt, that monkey viruses do cause cancer or other problems in humans. What they're concerned about is that monkey viruses were present in polio vaccines in the past and that no one knew, and that today monkeys are still being used to produce vaccines and it's still not known whether or not there are monkey viruses in them that you have not yet -- don't have the technology to detect. So parents are most interested in using vaccines that do not use monkeys for production. And this bring me to the question for Dr. Garcea. How many parents of young children with cancerous tumors that have SV40 in them, how many of these parents have been tested for the presence of SV40 in their bodies? DR. GARCEA: I don't quite know what you're asking. I mean, when we did the original study, we did not -- because of IRB regulations, decode and go back to the parents of these families and talk to them. So what you're asking is, subsequent to the study, have we analyzed other tumors that we've received because of this, and talked to the parents? Is that what you're saying? DR. LOWE-FISHER: No. Wouldn't it be interesting to know if, in these children -- these very young children who would not have received the vaccines that contained SV40 -- wouldn't it be interesting to know whether or not their parents are carrying SV40 -- DR. GARCEA: I think it would be a very interesting study, and as a part of a prospective study in looking at this, I think that that would be part of a sero-epidemiological study that you could do prospectively. But retrospectively, we can't do that, unfortunately, right now. DR. LOWE-FISHER: It would also be interesting to go back to the contaminated vaccines and PCR off the virus that's in them, and then compare the sequence that the sequence people are finding. DR. GARCEA: But let me just comment on your -- we can't do that because -- I would just mention, for the past seven years we have not had any money to do any of these experiments. DR. LOWE-FISHER: Well, we are hoping that this kind of research by independent scientists will get the funding it deserves because the public is most interested. And this is fine science and we're very interested in supporting that research. MODERATOR FRIED: Thank you. DR. LEDNICKY: Can I make a comment on that? This might be a little speculative but, the problem is it could also be that SV40 was always a human virus. It may have been in the human population a long time. And if you speculate and say, maybe SV40 was around much longer than JC or BK virus -- because lower primates predate humans -- maybe it's had a long time to adopt to humans. So it could be that it's in humans and just because some of us detect it in tumors, we need to prove that it's causing the tumors -- actually, one possible interpretation is, if someone has a tumor they might have an immune problem -- maybe immunosurveillance isn't cutting down an SV40 and other papomaviruses, and then so what we're detecting is circulating SV40. MR. KYLE: Could I comment, please? My name is Walter Kyle. I'm an attorney from Hingham, Massachusetts. I've been doing polio vaccine litigation for 25 years -- or 20 years, I should say -- primarily on behalf of plaintiffs. I know very little about genes; it's hard for me to distinguish them from a pair of levi's, but I do know that I have records going back into the late '50s, testimony before Congress, when Dr. Roderick Murray headed the Division of Biological Standards, in which he testified that no SV40 was ever found in inactivated polio vaccines. However, at the same time, in the same period of time at NIH in transcripts, Dr. Murray commented that it was entirely acceptable for SV40 to be present in SIV. I have both transcripts. Dr. Murray continued with this type of regulation of the polio vaccines. In 1968 after the discovery of the Marburg virus in the oral vaccine, he met with Lederle Labs and the same discussion was held. Yes, it's okay if you have these viruses in oral preparations because there's no evidence that it causes any harm. I think we've now come across the evidence that these viruses have caused harm. I think it was disingenuous for Dr. Murray to testify under oath in a prepared statement before Congress, something contrary to what he told his colleagues at NIH. I also would have a fundamental objection to the premise at this conference, that vaccines were clear of SV40 after 1963. You all know and I know, that every seed lot of Sabin vaccine is contaminated with SV40. What has occurred in the first production step is that you neutralize it with an anti-serum. Which leads you to the question of, how much is neutralized and how many people have gone back and looked at the harvest fluids in that first manufacturing step, to determine -- maybe if you had something creep through, something like JVC. The initial reports of progressive multifocal leukoencephalopathy found that two people that had it had only been exposed to oral polio vaccine. So to this day we have seed strains of the oral vaccine contaminated with SV40, and I don't think they changed their production methods in the early '60s for the IPV. There was no cutoff date, you didn't hear anybody at NIH come forward and say, we recalled that SV40 vaccine. It was not recalled. And I don't think anything was done. Murray testified before Congress that nothing needed to be done, because by the inactivation methods in effect at the time, that there was no SV40 out in the vaccine. And I don't think that's true, and the people that are familiar with this issue know that it's not true also. Dr. Shah pointed that out this morning. MODERATOR FRIED: Thank you for your thoughts. Ethel? DR. FANNING: I apologize for coming back to the papilloma viruses every time but I think maybe if some of you can learn a few things of all our traumatic experiences over the years, because we've gone through many of these discussions many years ago as well. We have looked at many archival specimens and what we see is that even -- it doesn't matter how these tumors were fixed; we have even degraded DNA going down to 100 base pairs -- but we can still amplify viral sequences up to 600/700 base pairs. Which means that these viruses are very, very resistant, and apparently polyoma is not much different. So that the method of fixation does not influence the stability of the virus. You still have viral particles in these tumors from which you can extract the DNA later on. So I tend to disagree with that point for the polyomaviruses. The other thing is about integration or episomal. In the cervical carcinomas that have been looked at, the majority of tumors have been looked at in the L1 region, also viral capsid protein, and in the majority of those tumors these viruses are integrated. And with the L1 primers the majority of the cervical carcinomas do contain papilloma virus DNA. So I think that should not make too much of a difference even if we don't know whether it's integrated in these tumors or not at this stage. And the third point that I just want to make is, if you're having a quality control, what you should maybe look at is where these tumors are coming from. If you're doing it from archival smears, how are you making those sections? Are you cleaning every little brush and are you using new blades between cutting every sample? It's not enough to just have an empty, sort of an odd slice in between. You have to clean everything from the beginning; the whole machine between tumors. The other thing is that we've had the experience that, in three cases we've received from three different clinics, batches of tumors which contained the same, sort of in the -- one batch would contain the same HPV type throughout the tumors, although they were completely different types of tumors. So in other words, in handling these tumors in the clinic, dividing it or sending it or packing it or whatever, it was contaminated during this process, and it was not contamination in the laboratory. So these are maybe things that one should keep in mind. MODERATOR FRIED: Thank you. John? DR. BERGSAGEL: John Bergsagel from Atlanta. I would agree with several of the panel members who have said that -- PCR in my opinion, doesn't prove anything. It's a screening method and you have to do something else to prove that what you found is what you think you found. But inasmuch as PCR is a useful technique for screening these extremely rare tumors, wouldn't it be useful to look at the animal models for these tumors if the real question is, whether SV40 causes these tumors, such as choroid plexus papillomas and carcinomas from hamsters and mice? DR. CARBONE: SV40 does cause exactly these tumors in hamsters. DR. BERGSAGEL: Yes, but if you use the exact same techniques, PCR amplification of fresh -- and even more importantly, formalin fixed and paraffin-embedded tumors from hamsters -- would you get the exact same results that we get from humans? DR. CARBONE: We used the hamsters as causative controls in our experiments, so the answer is yes. DR. BERGSAGEL: And the materials were handled in exactly the same way? DR. CARBONE: No. DR. BERGSAGEL: In other words, paraffin-embedded -- DR. CARBONE: No -- well, depends from what point. Obviously, the humans come from a surgery room, and the animals come from another route. DR. BERGSAGEL: Right, but if you took the animal's tumor and formalin fixed it and paraffin-embedded it to prepare your DNA as a control, and from multiple animals instead of just one which is known to be positive? DR. CARBONE: That's what we do. That's what we use. Of course, multiple animals -- a number of animals -- it depends what "multiple" means. But that's what we do. We use, for our experiment, hamster mesothelioma, SV40-induced hamster mesothelioma, and for our bone tumor experiment, SV40-induced hamster bone tumors. And we are very aware of the risk that there is when you use a microtome, when you're cutting this paraffin-embedded section and we certainly change blades, we change gloves, we clean everything, and then we start over again. That means, that takes a long time. MODERATOR FRIED: Okay. Go ahead. AUDIENCE PARTICIPANT: I think it's terribly, terribly important to stress for the two speakers before -- the lawyer and the representative of the public-at-large -- that not one of the speakers here today -- every one of them has been exquisitely careful not to claim causality. So please do not extract from what has been said, that it has been proven that SV40 is a cause of these tumors. MODERATOR FRIED: I think we all would agree with that. AUDIENCE PARTICIPANT: I'd just like to say something here. I was trying to be very careful to also say that the public is not as concerned about the fact whether or not you have proven beyond a shadow-of-a-doubt that there is causation. What they're concerned about is the fact that monkey viruses were in polio vaccine in the past; that we still perhaps, do not have the technology to totally guarantee they are not currently in the vaccines, and that they are concerned about the continued use of monkeys in the production of vaccines. So I just want to make clear that I wasn't implying that you had already come to the conclusion that there was causation. MODERATOR FRIED: Okay. Thank you very much. AUDIENCE PARTICIPANT: I'd also like to echo the comments of the former speaker here, that we haven't talked about causality. But I'd also like to emphasize what John Lednicky said, and as a person who deals with mesothelioma his points about basic immunosuppression are incredibly important. I mean, we know that these patients who are exposed to asbestos have T-cell subsets that just don't work well. We know that asbestos causes certain changes in their basic immune system that's going to make them functionally immunosuppressed. So I don't think we can say, wherever the T-antigen is from that that is it, that is complete. And I personally feel in dealing with these patients, that it is a complex intermix of whatever's going on, independent of the T-antigen situation, that the patients to begin with have some functional deficit. MODERATOR FRIED: Robin? DR. WEISS: We'll get on to causality tomorrow, but I have a question for Allen Gibbs. He told us this morning that he has more than a thousand -- MODERATOR FRIED: Could you speak into the microphone? DR. WEISS: Allen Gibbs mentioned this morning that he has more than a thousand mesotheliomas, this bountiful collection that Chris Wagner started. Do any of them go back earlier than 1955? DR. GIBBS: No, the earliest are 1960's, I think -- 1961/62, that sort of period. But I think there is a location where there may be a few that are pre-1960, and possibly back to 1955. MODERATOR FRIED: Because I think that's an important point, to look at things before the vaccines came about. DR. GIBBS: I'd just like to emphasize that I, being a pathologist, actually think that the archival material has a lot to tell us, and that's why we need to employ these techniques only on the archival material. And I understand what the concerns are and why there's an enthusiasm for using fresh material. But I think that if we all agree after a certain point in time, that the techniques are working and we are actually detecting SV40 virus, then it is important to exploit that archival material for the purposes of looking over different periods of time, and also looking at that proportion of mesotheliomas that we believe are reasonable evidence, and not asbestos-related. AUDIENCE PARTICIPANT: That brings me to my second question -- just one comment. A little concerned that we should not be matching historic and archival specimens with the controls that have been drawn from somewhat similar groups: age, sex, occupation, and perhaps most importantly, immunization history. That's the comment, and whether that's right you'll tell me. I'm glad that Allen raised the issue of non-asbestos versus asbestos. As far as I can tell, the mesos that have been discussed here have been entirely asbestos-related, or thought to be, is that correct? Anyone want to amplify that? DR. GIBBS: Certainly in my group that is the situation, but this was very much a pilot study and like Michele, we did this without any money, basically. And in terms of the controls, we did use pleural-based adenocarcinomas and non-malignant pleurae. The age ranges were similar but of course the mesotheliomas were dominated by asbestos exposure. But I think that's a study further down the line. DR. GOEDERT: Jim Goedert from NCI. Howard Strickler and I have discussed a number of different epidemiologic studies and to answer Robin's question, there are in fact, resources of specimens from pre-1955 from the U.S. Armed Forces Institute of Pathology that we can delve into. But you know, our priority was to try and come up with an adequately-sensitive and specific and reproducible assay before trying to delve into those specific epidemiologic questions. But I think the materials ought to be available and the controls, obviously, are critical in terms of how they would be matched. MODERATOR FRIED: So they could be sent out to people here on the panel? Yes? DR. RATNER: I'm Herbert Ratner, the former Health Officer of Oak Park, Illinois, and the announcement was made April the 12, 1955, Tommy Vance has reported that the vaccine was safe and effective. And within a few days the National Foundation had this vaccine -- I won't go into the past history of that vaccine -- but it was delivered throughout the United States so that every 1st and 2nd grader, as a free gift of that vaccine -- every 1st and 2nd grader -- and in the next week or two, that vaccine was given to every 1st and 2nd grader. I think Oak Park was probably the only one who decided to sit down that free gift, vaccination gift, just to see how things were going along. There were other reasons, too. But I decided that before parents signed an authorization slip, which makes it possible to get the vaccine, that I should make available to them -- which I did in 11 talks that week -- be willing to answer questions that they had in terms of the risk of polio that summer, etc. By just taking a neutral position at that time, you had all the pressure from the Foundation to get that vaccine going because of an impending summer polio epidemic -- the usual summer epidemic -- and that was the only thought in people's minds: how fast, how well do mothers love their children? They didn't rush to get the vaccine, and things like that. And in the midst of my talks -- I had two days of my talks -- my community got very upset that where everybody else was giving the vaccine, we were holding out. And it caused quite a consternation in the Chicago area. It got to the science -- Art Snider who was the Science writer for one of the major newspapers -- he said Herb, what's going on there? I said, well come out and listen to my talk, etc. I have the talk on Tuesday and Wednesday he called me up and said, you're more right than you know. Because they just got the first report of the Cutter vaccine situation where six cases in San Francisco and one in Chicago area, both from the same manufacturer, both from the same lot number, and we were in consternation three. I had to postpone -- actually, I was about the only one in the country that was in a position of not having anybody in my community immunized, and so I could sit it out. And I made one appointment to use the vaccine, to give that, give to their parents -- one week later or two weeks later, whatever it was -- and after that, the Cutter situation got worse. And the local paper, as a result, had a story, checked around, in which they thought I had a very unique opinion that I hadn't given the vaccine. MODERATOR FRIED: I think we're going to discuss the vaccines more tomorrow. I mean, this is mainly for the techniques, so -- DR. RATNER: Can I have about a minute more? MODERATOR FRIED: One minute. DR. RATNER: Yes. Keep up my same thought. The day that the local paper came out with the backing of all of the -- everybody in the community, kind of -- Seeley, the Surgeon General, called up the program because he wanted to make a safe vaccine safer was his exact terms. They had to stop that thing because of the difficulty of the vaccine. And if all of you knew the difficulties they had with the Salk vaccine, whose position on inactivation turned out to be false -- universally accepted as false -- and how they kept packing it up and packing it up and packing it up, and they had to keep the program going and going. But I'm telling you that every 1st and 2nd grade child in the United States, which represented about 85 or 90 percent, got a vaccine which had live polio viruses in it, definitely established, and at that time they found out that the SV40 was -- MODERATOR FRIED: I -- DR. RATNER: Just one sentence, please. That the SV40 was not activated, and so that meant that there was SV40 in all of the vaccines around the country, and that was confirmed by -- this is my last sentence -- that was confirmed by anybody who focused epidemiologically. There were cases popping up all over the States -- and this was confirmed by the German Health Ministry who were doing the same thing in Germany -- that polio virus was being distributed. And if you people could see -- MODERATOR FRIED: I think I have to stop you, because we -- DR. RATNER: Could I just have a half-a-sentence? MODERATOR FRIED: You've had a half-a-sentence. DR. RATNER: If you people sit here and say that the vaccine didn't pass on polio or SV40, you don't know what happened in those times. And I'm talking about 1955, for the next ten years or more. It's strange to me, as an epidemiologist working right on the field, to hear people somehow deny the vaccine -- one more sentence, please. Harry Francis was attacked right after his report -- MODERATOR FRIED: I think -- why don't you save this for tomorrow? DR. RATNER: Okay. DR. URNOVITZ: Hi, I'm Howard Urnovitz and I'm from Berkeley. That's the other coast. First let me thank the -- I want to say thank you to the FDA, NIH. I think it's a brave move to have us all come together; I think it's very productive. I think everybody's going to work out false positive problems. You just send samples to each other and I think that's not going to be a problem. And Dr. Carbone shouldn't get rattled. There's a lot of us who believe that what you've done is a breakthrough, and most of you here, we're very excited about it. I want to make a comment that the chimera thing is of interest to me; that Dr. Frisque had said with the SV40 and JC virus. Is that there were dozens of viruses in those preparations. I think it's -- this is an important first step to talk about SV40 because we know a lot about them and we could start this as a springboard. I don't think anybody here would walk away saying there's a cause of cancer. It's probably multifactorial and we're looking at the components. The question to the panel is, as you go forward building your primers and as you see there are certain primers well lighted up, is to be mindful of the fact that some of those might be other types of hybrids. Certainly we know about SV40 adenovirus, but there were also coxacky and other adenoviruses in there, there were herpes viruses in those preparations. There may have been chimeras and those in themselves might be important too. So as you do your primer sets, has anybody looked at doing multiplex as the screen and then sequencing as the verification? MODERATOR FRIED: Anybody want to take that? Janet? DR. BUTEL: We haven't done that. MODERATOR FRIED: Okay. I think we've run out of time. We've had a very fruitful and interesting discussion. I think people would agree that the techniques are getting down to detecting things now and maybe we can get a coded test panel of cells to go to the different people interested. We obviously need the finances for this, and maybe people should be doing PCR of the vaccines to see exactly what strains were in that and how they match up to what people are finding; whether there's really an endogenous virus or it came from somewhere else. Okay, thank you very much to all the panel members. (Whereupon, the foregoing matter went off the record at 3:55 p.m. and went back on the record at 4:20 p.m.) CHAIRMAN SNIDER: We're ready to start this last session. We're at perhaps, the most difficult part of the day, but I think one of the most important parts of the day. This session is on human exposure to SV40. My name is Dixie Snider; I'm the Associate Director for Science at The Centers for Disease Control and Prevention in Atlanta. We too, are happy to be co-sponsoring this meeting and look forward to the rest of the meeting and to deliberating on the significance of the outcomes. Our first speaker for this session is well-known to everyone in the vaccine field. It's Dr. Maurice Hilleman who is at the Merck Institute for Therapeutic Research. Dr. Hilleman. DR. HILLEMAN: Well thank you, Dr. Snider. Having been there, perhaps I can recite the history. The development of both killed and live poliomyelitis virus vaccines was at the pioneering forefront of what was to become a new golden age of vaccinology. For polio virus vaccines, new technologies needed to be conceived and developed, and as might be expected, there were significant challenges which related mainly to whether the polio virus in killed vaccines was completely inactivated by formaldehyde and whether the virus in live virus vaccines was underattenuated and caused poliomyelitis in human beings. Adding to these complexities, both kinds of vaccines depended upon polio virus propagation and Maitland-type minced renal tissues of monkeys, or in cell cultures of monkey kidney. Both cultures, it was later to be determined, were commonly infected with any of more than 40 different indigenous viruses of monkeys. The most commonly used monkeys were the Macacus rhesus and the Macacus cynomolgus species. Now, as part of the requirements for killed polio virus vaccines promulgated by the NIH's Division of Biologic Standards, later named the Bureau of Biologics, it was necessary to demonstrate the inactivation of all detectable viruses. Live polio virus vaccine, by contrast, followed different rules that required the cell cultures to be free of known viruses from the start. All manufacturers who distributed polio vaccine in the United States were required to meet the U.S. standards. Well, the prevalence of contagious viral infections in Macacus monkeys was vastly amplified by the shipping and caging conditions which were standard at the time, including necessary contact between animals which occurred during transport, on holding at airports, or on housing at the final destination. The modes of viral transmission between monkeys were possibly by the respiratory route or by ingestion of monkey urine or maybe even feces containing the agent. Well, the discovery of SV40 virus was born of change and serendipity. An urgent need for monkeys for research and development of other live virus vaccines led to a search for monkeys with as few wild virus infections as possible. This caused the speaker to consult Dr. William Mann who was then Director of the National Zoological Park in Washington, D.C., for advice on how to capture and transport monkeys with the least chance for virus exposure. Well, Dr. Mann advised that African Green monkeys, that is, cercopithecus aethiops, could be caught in West Africa, transloaded at Madrid where there was no traffic and non-human primates, and then transported to New York and on to our laboratories. Heeding Dr. Mann's advice, these monkeys were obtained and they provided a source for kidneys. Well, most surprising, the cercopithecus cultures showed remarkable capability for propagation with cytopathic change of a little of a hitherto unknown, indigenous, Macacus virus that was otherwise undetectable at that particular time. Now, this virus was noted to produce vacuolar, cytopathic changes in the cytoplasm of cercopithecus renal cultures in culture. It was called a vacuolating agent and it was later renamed simian virus 40, or SV40. Preliminary findings were presented at the June 1960 meeting of the Second International Live Poliomyelitis Vaccine Conference, which was held under the sponsorship of the Sister Elizabeth Kennedy Foundation, at the Pan American Health Organization headquartered in Washington, D.C. Thereafter, studies of the SV40 virus were continued, both in our laboratories and elsewhere. The SV40 virus was reported at the meeting to be a hitherto, unknown agent whose small size -- and was cytopathic for cercopithecus kidney cells. By contrast, it caused only an inapparent, non-cytopathic infection in primary Macacus kidney, and in primary or continuous passage human cells. All isolates that were examined were antigenically homogeneous as determined in serum-neutralization tests. It was reported at the June 1960, meeting, that cercopithecus kidney cells in culture were nearly always free of SV40 virus, but cultures of Macacus monkey kidneys, Sabin live polio viruses, and seed stocks of viruses used to prepare experimental killed adenovirus vaccine were found to contain the virus. At the same meeting it was reported that cercopithecus monkeys were free of SV40 antibody, but that sera from most Macacus monkeys were positive. More than half of all the sera from the human recipients included in our study who had received killed Salk or adenovirus vaccines that had been prepared using virus grown in Macacus cell cultures, were positive. The antiviral antibodies that were demonstrated in the sera of recipients of the killed Salk and adenovirus vaccines were appropriately interpreted as having been induced by the inactivated SV40 virus that was present in the preparations. Recipients of Sabin vaccine -- that's the live vaccine -- were devoid of antibody even though it was shown later by others that the SV40 virus infects the human gut and is excreted in the feces, with probably lack however, of significant, systemic viral infection. Well, at the time of that June meeting the vacuolating virus appeared to be of essentially universal presence in Macacus Rhesus monkey kidney cell cultures, frequently present in Macacus cynomolgus kidney cultures, and relatively rare in African Green monkey kidney cultures. The new virus appeared different from other known monkey viruses such as those described by Hull, because of the distinctive vacuolating type of cytopathic change seen in infected cercopithecus kidney cell cultures. Failure of the vacuolating virus to cause cytopathic changes in Rhesus or cynomolgus monkey kidney cell cultures was a hallmark for the vacuolating agent. Resistance of the virus to ether and failure of hemagglutination and hemabsorption such as shown by the mix of viruses were also distinguishing characteristics. The vacuolating virus appeared to be just one more of the troublesome simian agents to be screened for and eliminated from, virus seed stocks and from live virus vaccines. Lack of antibody response in human subjects who were fed live polio vaccines containing the vacuolating agent, suggested the lack of substantive proliferations of this semi-permissive virus in the human being under the conditions employed. Well, discovery of the SV40 virus was possible only after a cell culture system was available that would detect its presence. And that's important. The detection in Green Monkey kidney culture of this inapparent virus infection of Rhesus and cynomolgus monkey kidneys represented the first instance of demonstration of a non-detectible, indigenous monkey virus using a monkey renal cell culture. Then in September of 1960 the inactivation kinetics of the vacuolating virus using one to 4,000 formalin at 37 degrees -- the conditions used to inactivate polio virus vaccine -- were described. Inactivation of SV40 virus having a rate constant similar to that of poliomyelitis virus, was observed. Under the conditions used in this study, our testing indicated that the vacuolating virus was destroyed during the polio virus inactivation process. The optimal solution to the live virus vaccine problem however, appeared to lie in total elimination of the virus from the production system as soon as possible. In 1961, when SV40 virus of higher infectivity titer was available, and when more sensitive tests for its detection were developed, a new and unique pattern for its formaldehyde inactivation kinets were found, as shown here in red. These studies disclosed an asymptotic relationship in the inactivation curve after about 99.99/100th's percent -- that would be 4 logs to the base of 10 -- of the virus had been killed. Virus that was subcultured from the plateau portion of the curve showed the same inactivation pattern as the original. Just why approximately one in 10,000 SV40 virus particles are refractory to inactivation by formaldehyde has been an enigma for more than three decades. It is now known, however, that the closed, double-stranded circle of the SV40 viral DNA genome is super coiled, but that a single break or a nick in one strand of the double strand gives a relaxed ring. A double break gives a linear double strand. Well, completely double-stranded DNA provides no exposure of immuno or amino groups with which formaldehyde can react. This might give an explanation for the means by which the chance presence of a single, resistant virus particle in every 10,000 SV40 virus particles can escape inactivation. Reports to the Division of Biologic Standards of survival of this very small fraction of SV40 virus, led the Division to require demonstration of freedom from detectable, live virus -- SV40 live virus -- in the final product when a volume of 500 doses of finished vaccine per lot was tested in cercopithecus renal cell cultures. As part of our studies to characterize SV40 virus, newborn hamsters had been inoculated subcutaneously and intracerebrally with live SV40 virus to test for possible oncogenicity such as had been shown for SE polyomavirus of mice. Hamsters that are less than 24 hours of age have a relatively deficient immune system and provide an in vivo animal model to study viral oncogenesis; albeit, without it having any known or established relevance to the human species. And I would emphasize that. In the test, mildly invasive fibromatous tumors appeared after five to ten months in nearly all hamsters given massive doses of SV40 virus. Now, this was 320,000 50 percent tissue culture, infectious doses per hamster -- a huge dose. Tumors did not appear in appropriate placebo controls. The tumors were transplantable to new animals and markers for SV40 virus were shown present in the tumors by specific virus recovery and by immunofluorescent identification of the T-antigen. Well, it's notable that SV40 virus tumorigenicity in hamsters is highly dose-dependent, and that no tumors appeared following injection of less than 1,000 tissue culture doses of the virus. It was shown also that SV40 virus tumor appearance was highly diminished when non-replicable, whole, cobalt-irradiated SV40 tumor cells were given prior to or as late as, 76 days following injection of the homologous virus into newborn hamsters. This anti-cancer vaccine proved to be both prophylactic and therapeutic. It was a new principle. The appearance of tumors in hamsters inoculated with SV40 virus gave an explanation for the findings by Eddy that injection of extracts of ground, primary cell cultures of Macacus monkey kidney-induced tumors in newborn hamsters. For want of detection of any oncogenic stimulator, Eddy referred to the tumor-inducing entity as an oncogenic substance. In a later publication, after SV40 had been discovered, Eddy reported isolation of SV40 virus in cercopithecus cells from the same monkey kidney preparations used in our earlier study. Well, while the studies at Merck were in progress, the early results of the neonatal hamster tumorigenicity tests were reported by us to the division of biologic standards and in turn, to the technical committee on poliomyelitis vaccine. This committee was a group of leading scientists who served as polio virus vaccine advisors to the U.S. Public Health Service. The division and the committee had previously received reports of possible, live, SV40 virus in commercial, killed, polio virus vaccine. The view of both the division and the technical committee was that no untoward effects in human subjects could be attributed to the agent. They also concluded that there was no evidence that the small amount -- very small amount -- of live, SV40 virus which also was subsequently determined to be only semi-permissive for man, was capable of producing disease in human beings when introduced subcutaneously or intramuscularly in a formalinized vaccine. Further, the committee stated that although the presence of the vacuolating virus in the killed vaccine does not prevent the development of immunity against polio in vaccinated persons. The elimination during the process of manufacturing polio vaccine would constitute another step in the continued improvement in the potency and the purity of the product. Well, by late summer of 1962, the Division of Biologic Standards recommended that all pools of polio virus and adenovirus be shown free of SV40 prior to the addition of formaldehyde. SV40 virus-free pools were made a requirement early in 1963, but by that time, you know, all three serotypes of Sabin live polio vaccine had been licensed by the Division for use in the United States. The Sabin live virus vaccine was readily accepted by the physicians and public health practitioners because of the simplicity by which it could be administered orally. Salk vaccine use was diminished and it almost disappeared. Well, now in closing, I think it's worthy to note that within a relatively short period of time following the discovery of SV40 virus, the agent had been found present in poliomyelitis vaccines, it had been shown to be incompletely inactivated by formaldehyde, and had been shown to be oncogenic when tested in newborn hamsters. In another short time period, the methodologies for excluding SV40 virus were developed, validated, and ultimately utilized. And it was of importance that during the time period prior to licensure of the live Sabin vaccine, the Division of Biologic Standards had been able to clear sufficient Salk vaccine for distribution to allow the large poliomyelitis immunization campaign in the U.S.A. to continue without interruption. Because of this, thousands of cases of poliomyelitis that would otherwise have occurred, were averted. Thank you. CHAIRMAN SNIDER: Thank you very much, Dr. Hilleman for that excellent background and historical perspective. Our next speaker is Dr. Frank O'Neill from the VA Medical Center, Salt Lake City, Utah, who will speak on the host range analysis of SV40 and SV40/BK hybrid genomes and virus latency. Dr. O'Neill. DR. O'NEILL: First I'd like to thank Dr. Lewis and all the meeting organizers for inviting me to this meeting. One of the projects in my laboratory over the last several years has been an analysis of SV40 growth in a variety of human cell types. And we've tried to determine which cell types SV40 grows well in, and which cell types it does not. And in those cell types where SV40 grows poorly or slowly, what about the virus is causing this slow growth? And I'd like to summarize our findings in the following points. One is that SV40 grows well in some human cells types. In cell types which it does not grow well in, like fibroblast and human embryonic kidney cells, this slow growth appears to be caused by some function of the SV40 late region, because when we replace the SV40 late region with the late region from BK virus or RF virus -- a variant of BK -- we now get rapid growth in human embryonic kidney cells and in fibroblast. Finally, in fibroblasts then, in human embryonic kidney cells, wild type SV40 produces very small amounts of T-antigen but it produces very large amounts of the capsid protein, VP1. And in fact, there may be 150 times more VP1 than there is T-antigen. And this overexpression of the VP1 gene, or the late region, appears to inhibit T-antigen production. So this is an outline of the talk. There are three kinds of theoretical growth patterns of SV40 in cells: semi-permissive cells where there's very slow growth and not much virus produced -- and very little cell killing also; fully permissive cells like simian cells, CV1 monkey cells which virus grows rapidly and it kills almost all the cells; and there may be totally non-permissive cells. There may be some human cell types that are totally non-permissible. There may be very little T-antigen expression that has been reported previously, but I'd like to qualify that and say that, in some of these studies that showed no T-antigen production in human embryonic kidney cells and in fibroblast, a lot of those plasmids had the viral DNA still covalently linked to the plasmid. And we've shown recently that plasma DNA strongly interferes with the expression of the T-antigen gene in human cells. And as I mentioned earlier, point 3 here, the mechanisms of growth for slow growth in human embryonic kidney cells, appears to be the SV40 late region. And I also have some experiments about viral latency but it's highly unlikely I'll have time to get into that. So these are some of the features that are the growth of SV40 in human cells: human embryonic kidney cells and fibroblasts. Only about 20 percent of the cells initially appear to be infected. And as I mentioned, little T-antigen is produced and ultimately the cells become morphologically transformed. So we went on and started to analyze a variety of human cells types to see if SV40 would grow in other cell types besides fibroblasts and human embryonic kidney cells. And you can see on the first line we have monkey kidney cells, BSE1, TC7, and CV1s, and growth is optimal in those cell types. But as I mentioned, HFF fibroblasts and HEK cells, the virus grows poorly or slowly. But then we looked at some neural cells, neuroblastomas; the virus seemed to grow fairly well. But after a couple of rounds of the replication cycle of the virus, the cells become resistant. In two glioblastomas, A172 and A182, SV40 seems to grow quite well. In a lung cancer cell line, AT357, SV40 grows very well. It grows as well in those cells as it does in simian cells. And in two rhabdomyosarcoma cell lines, again, SV40 grows very well. And one renal carcinoma cell line, SV40 grows quite well also. So there's a variety of human tumors that support lytic infection by SV40. And on the second line you'll see fetal brain cells. Fetal brain cells that are rich in spongioblasts support rapid growth by SV40. SV40 grows as well in those cells as it does in Green Monkey kidney cells. Now, one of the things that has been indicated previously, that in human embryonic kidney cells and fibroblast there is very poor growth of SV40. And we agree with that unless you let the cells -- unless you maintain the cell cultures for long periods of time. If you harvest the cells to extract viral DNA within a week to three weeks after infection, you find very little DNA, and those experiments appear in lanes 3, 4, and 5. Lane 1 is the amount of DNA that you would see classically, after extraction of infected monkey cells. But if you let the human cells that have been infected, you maintain them in culture for at least six weeks, you then see a lot of viral DNA. And that's Lane 6. There's as much viral DNA from those cells as you would get in a lytic infection of monkey cells. In lanes 7, 8, and 9 are BK virus infection of human embryonic kidney cells or fibroblasts, and BK virus of course, grows well in both of those cell types -- those cell types that allow SV40 to grow only slowly. So SV40 will grow in these so-called semi-permissive cells. If you wait long enough you can observe that good growth. Now, one of the things that I want to mention about SV40 growth and neural cells, like spongioblast and some glioblastomas, is that the virus makes a lot of mistakes. When you isolate the DNA, even after you've started an infection with plat purified virus or molecularly cloned viral DNA, you see a lot of defective, interfering viral DNA particles. And when you analyze even those particles that don't seem to be defective, you can see a lot of mutations. You see rearrangements in the regulatory region, in the 72 base pair repeats, and in sequences between the 72 base pair repeats and the beginning of the VP2 gene. We also see mutations, deletions, base substitutions and insertions at the 3-prime end of the T-antigen gene, and the 3-prime end of the VP1 gene. Another thing that we see in neural cells is that the viral DNA seems to split. Instead of having all the viral sequences necessary for an infection in one molecule, we see the viral DNA sequences split into two, complementing, defective molecules -- like on this slide. And the circle on the left, that's a genome that contains just a T-antigen gene; the late region has been deleted. On the genome circle on the right we see the late region that has all the capsid genes, but the T-antigen gene has been deleted from that. Both of these molecules, when introduced together, will produced a lytic infection, and they have the same host range as wild type SV40. Now, there are similar viruses that have been described for JC and for BK. Two BK variants called RF and MG, have the same genome organization and they show this genome organization isolated directly from the patients. So one of the things that we wanted to do was determine what causes slow growth of SV40 in fibroblast and human embryonic kidney cells? And we know that in those cell types, BK and the RF variant of BK grow quite well. So what we did was, make reassortments of viral genomes using early SV40 and late RF, or early RF and late SV40; a variety of combinations between SV40 and BK or SV40 and JC. And what we found initially was that every time we had the SV40 late region complementing BK or JC, virus growth was very poor, very slow. But in cases where we had late BK complementing early SV40, virus growth was rapid. Those hybrid viruses appeared to grow almost as well as BK or RF did in human fibroblast and kidney cells. So that suggested that there was something in the SV40 late region which was restricting growth. What we found when we do this experiment -- if you will assume that that left circle is early SV40 and the right circle is late RF -- what we find is that there's always recombination between SV40 and RF such that the RF genome acquires an SV40 regulatory region, and that always happens every time we do the experiment. One of these variants of late RF that has an SV40 regulatory region is called clone-H. and we decided to determine if clone-H could stimulate the growth of wild type SV40 in human fibroblast. So we introduced both clone-H and wild type SV40 -- and that's a map for wild type SV40 -- and to human fibroblast. So what we did is, we introduced both viral genomes into human fibroblasts and the virus growth was very slow. But when we analyzed the viral DNA after the first passage, we could see very little wild type SV40 DNA. When we took that lysate and passed it several more times, the wild type SV40 DNA had totally disappeared and it was replaced by a variant of SV40 that had only the early region in it; the late region was deleted. So late RF could not help -- late RF clone-H could not help wild type SV40 grow in human cells. The SV that did grow had lost the late region, suggesting that there was something in the late region that had some cisinhibitory effect. So further evidence that something in the late region was inhibitory to growth. The next thing we did -- so in lane 1 you can see the bottom band is late RF clone-H as to one passage. And lane 2 is after three passages, and that bright band is early SV40 that has lost the SV40 late region and it's now complemented by late RF clone-H. So then we decided to ligate the late RF sequence to the early SV40 sequence to make a hybrid genome or a chimeric genome that had both DNAs in one circle. And that's shown in the bottom circle in this slide. And that virus, or that viral DNA, has the same phenotype as the other hybrids I've described. This virus now grows in human cells and it also grows in monkey cells. So this virus with a chimeric genome grows as well in monkey kidney cells as it does in human embryonic kidney cells. So again, it looks like there's something in the SV40 late region which restricts growth in fibroblasts and in kidney cells. So the next thing I'd like to address is, what do the proteins look like after you transfect or infect human cells with wild type SV40, early SV40 which has a deleted late region, or the chimeric genome? And if you look at lanes 1 and 2, that's early SV40 DNA minus the late region in human cells for day 3 or day 6 in lane 2, and you see there's a fair amount of T-antigen. In lanes 3 and 4 is wild type SV40 and you see there's very little T-antigen at day 3, and at day 6 it's almost undetectable. But if you look at the bottom of those lanes you'll see plenty of VP1. A lot more VP1 than T-antigen. In human embryonic kidney cells you get a similar result. You get plenty of T-antigen with just the early regions but very little T-antigen when you use wild type, but also a lot of VP1. So VP1, the late region appears to be overexpressed compared to T-antigen, and you can show that in northern blots. When you use the chimeric genome, T-antigen is poorly expressed early, but after a few days you see plenty of T-antigen. Again, the late region is overexpressed. And the same results appear on this slide, but in addition we show what kind of amounts of T-antigen are produced in monkey cells with early SV40 and with wild type SV40. Again, you can see that when the late region is present you get lots of VP1 and you inhibit expression of the T-antigen gene, so you get less T-antigen. In the human cells, at days 3 and 6 and 10, you can see that with wild type, T-antigen starts to fall off, as it does also with early SV40. With wild type, after day 10 you start to see the reappearance of T-antigen and also more VP1. So that by about six weeks after infection when the maximum amounts of viral DNA are present and almost all the cells are T-antigen positive, you see huge amounts of VP1 but still very small amounts of T-antigen. Much less T-antigen; there's about 150 times more VP1 than there is T-antigen. In monkey cells as the infection progresses, you see more and mo |