Blood Products Advisory Committee Meeting Announcement (BPAC) December 14-15, 2010

[George]

Can anyone explain why each lab used a different type of test?

Can anyone explain why we are down to three labs now? We started with the WPI, the CDC, Two FDA labs, a NIH lab, and the NCI (Ruscetti) lab. We seem to be down to the WPI, the CDC, NCI and Gen-Probe. Any takers on that question?

 

[Cort]

No results from the WPI????

Gen-Probe takes a shot using a different methodology - and finds nothing - but its using new technology that has not been validated, so it's a bit of a wash...

Phase IIb Gen-Probe results

Target capture/Transcription-Mediated Amplification (TMA)/chemilumenescent detection

-Platform: TIGRIS system
-High throughput, fully automated NAT system
-Currently used routinely world-wide for NAT blood screening

-Assay design: duplex assay that targets conserved sequences in 2 separate regions of XMRV genome
-Ability to detect MLV-like sequences unknown; new assay to detect a broad range of MLV sequences under design
-Each reaction includes an internal control which validates assay steps (target capture, amplification and detection)

-Sample volumes: 500 ul plasma, 50 ul WB or 100 ul PBMC

All plasma and PBMCs samples from both days were negative

 

[Cort]

Here's WPI

Remember samples are blinded and from someone who tested positively and this is newly drawn, never before stored blood at the WPI

They wrongly identified a negative control as positive - but think they can explain that..

They were able to identify XMRV correctly in 3 of 4 positives - it wasn't easy, they could only do so on one day and they missed on one but the important thing is that by and large they identified the positive samples correctly - a big win

This suggests a) because the controls tested negative, their lab is not contaminating the samples
b) while its not always easy to find XMRV they can find it and in mostly the right samples but not always....

On the other hand if they had only 5 samples - they would have known 4 should have been positive - so much for blinding...and on one of the days they misidentified which one was positive.

It's a little sketchy because they missed on one control and XMRV is not easy to find - and, suggests that really, no one has a really good test for it yet.

The salient fact is though, that, by and large the WPI picked out the controls from the positives and that is the acid test. It suggests to me that the WPI is doing some right the other labs are not and that the BWG should be very carefully going over the labs procedures - down to the smallest detail - to see what the WPI is doing differently.

To me this seems this is something like a weak win for the WPI. If they had gotten that negative right it would have been bigger.

If you put this in a chart you find that they picked the right positive only 2/5 times..They got the negative right 1/2 times....They didn't even mention in this chart what happened to samples 2,3,4 on Day 0!

The straight PCR test needs alot of work - which is why the WPI is now focusing on culturing I suppose.

Phase IIb WPI results

Nested RT-PCR for MRV gag followed by sequencing of positive bands to confirm specificity

-RNA extracted directly from plasma

-Total nucleic acid extracted from PBMC and RT step performed

WB testing has not been completed (error discovered)

PBMC results:

-For Day 0, Subject 1 and the pedigreed negative control were positive (note on slide: investigation following decoding of results determined that there was a procedural error during PBMC sample extraction involving reuse of needles, employed to lyse cells and shear DNA, on sequential PBMC cell pellets.)

-For Day 2 only, Subjects 1 and 4 were negative as well as the pedigreed negative control
-For Day 2 only, Subjects 2 and 3 were positive

Plasma results: All subjects and the control were negative for both days

 

[CBS]

One of the questions I have and hope we get an answer to tomorrow is whether NCI/DRP is different from NCI/Ruscetti (on a later slide re: serology results).

So confusing STILL!

Val,

First, Thank you for being our eyes and ears on the 14th. You really came through for everyone.

The Ruscetti Lab is in NCI's Center for Cancer Research -
NCI-Frederick
Leukocyte Biology Section
Building 567

http://ccr.cancer.gov/Staff/staff.asp?profileid=5488

The NCI's HIV Drug Resistance Program (DRP) located on the Frederick campus but it is located in a different building:

[FONT=Geneva,Arial,Helvetica,Swiss,SunSans-Regular]HIV Drug Resistance Program
National Cancer Institute
NCI-Frederick, Building 535, Room 109

Here's a link to the HIV-DRP research teams: http://home.ncifcrf.gov/hivdrp/research_team.html

So, it appears that Ruscetti's lab is separate from the DRP. However, it would be interesting to know if they collaborated in any manner.


ETA: Sandra Ruscetti's lab also appears to be seperate from the DRP:

[/FONT]Laboratory of Cancer Prevention
Head, Retroviral Pathogenesis Section Senior Investigator

Building 567, Room 152
NCI-Frederick
[FONT=Geneva,Arial,Helvetica,Swiss,SunSans-Regular]
http://ccr.cancer.gov/Staff/staff.asp?profileid=5518
[/FONT]

 

[George]

CBS, is this what Dr. Mikovits meant when she said they(NCI) "took it to a new lab that had not done any previous XMRV testing" ?

 

[Cort]

Using NAT only WPI finds positives and not all the time but remarkably day 2 shows up again. This is a really good sign because a pattern suggests something real - something biological - is going on...

(on the other hand could it mean that the longer it is in the WPI the more likely it is to pick up something? Remember, though, the CDC also had a day 2 pattern in Phase IIa....suggesting the pattern is biological- something to do with the virus)

Interestingly WPI again on Day 1 finds 'the negative control' (one negative control!) positive...which again is a little helpful..something is happening on Day 1..

I think the Day 2 pattern is really going to help them.....

Summary of Phase IIb NAT Results (This is a big table, so I will summarize it)

Using NAT, 3 of the labs had all negatives for both plasma and PBMC for all subjects and the negative control, on both days. The labs were NCI/DRP, Gen-Pro and the CDC.

Using NAT, WPI found positives for
-Subject 1 from Day 0 with PBMC only
-Subject 2 from Day 2 with PBMC only
-Subject 3 from Day 2 with PBMC only
-Negative control was positive on Day 0 with PBMC only

 

[Cort]

CBS, is this what Dr. Mikovits meant when she said they(NCI) "took it to a new lab that had not done any previous XMRV testing" ?

I think that was the UK study?

 

[Cort]

The all important antibody tests

A real mixture -suggesting that the serology REALLY tests need work...

The NCI identifies the positives 5/8 times correctly and the control half the time. Which means they were incorrect about 40% and 50% of the time - which means too many false negatives and positives... and too little testing as well (just 5 samples).

If the WPI accords with these findings their antibody tests are not ready for game time - unless they test them again and again and decide that only the positives are correct. If that's right they could say, yes, we picked up 75% of the positives.

The problem is that we don't know about the other side - the false positives. Finding the control positive suggests they are picking up those as well.

I can't imagine you can tell much with 5 samples anyway...particularly just one control sample???

CDC finds nothing. They reported they looked for multiple MLV's - so they are looking for the pMLV's and found nothing

Summary of Phase IIb Serology Results (so far)

CDC results all negative, all subjects and control, both days
-Used Western blot for multiple MuLVs (Switzer, et al, 2010)

NCI/Ruscetti results

-Subject 1 positive on Day 0 only
-Subject 2 negative both days
-Subject 3 positive both days
-Subject 4 positive both days
-Negative control positive on Day 2

-Used flow cytometry on cells expressing spleen focus-forming virus env (Lombardi, et al, 2009)

Note: All tests on plasma

Comment: The presenter stated that only 2 labs' results were presented because others had not yet completed testing. Remember that later on in the meeting, Judy reported that WPI's serology results were concordant with NCI's (but data not presented).

 

[CBS]

CBS, is this what Dr. Mikovits meant when she said they(NCI) "took it to a new lab that had not done any previous XMRV testing" ?

I was not aware of her having said that but it would explain that statement.

 

[Cort]

Conclusions from Phase IIb

In this round of testing, only 1/4 labs detected XMRV in clinical samples and only on PBMC (me-this conclusion is confusing because the previous slides sure look like there were positives found from more than 1 lab???)

Ultracentrifugation or direct extraction of plasma did not seem to make a difference in detection by NCI lab.

The sensitive NAT assay from a diagnostic company unable to detect

I think only the WPI found positives in the second round collected using the phlebotomy company.... That could be a danger sign for the WPI - except that, by and large, they identified the positives correctly.......

So it's more of a danger sign for everyone else, I think :Retro smile::Retro smile:

 

[Cort]

Overall Conclusions from Phase II

Their conclusion - include more samples - a good idea!

All in all I can see why the WPI would not be displeased by these results. XMRV is a tough nut to crack even for them but they seem closer than anyone to cracking it. Dr. Mikovits is now going to talk on XMRV, Lyme, atypical MS, etc...... I think she feels good.

What is the Phase III Panel?

Based on Phase II findings, no clear advantage to delayed processing
(me-I don't get this conclusion either. Seemed like Day 2 testing was a bit better overall, but agree results weren't a lot better.)

Will include serology in parallel with NAT in future studies

Continue with collection of Phase III panel
-Include more positive and negative samples and differently sourced samples
-PBMC, WB and plasma will be used, with a standard next-day processing protocol

 

[CBS]

My left nut

I would like to see detection in blood samples compared to detection in tissue samples.

If I recall correctly, the animal model study data detected XMRV in the testes during both the "acute" and "chronic" phases of infection. Add to that the findings of Ila Singh (as per the patent application) that:

[0108] XMRV present in other tissue and cancer types. Tissues from approximately 80 autopsies on males aged 18 to 85 years were analyzed for the presence of XMRV using the described methods. Subjects included eight individuals with prostate cancer. Of these eight, two had XMRV present in their prostate cancer, in agreement with earlier results showing that approximately 25% of prostate cancers have detectable XMRV. Tissues from every available organ from the eight subjects with prostate cancer were also analyzed for the presence of XMRV. In these subjects, the only other tissue where XMRV was found to be present was the Leydig cells in the testes, the cells that produce testosterone (Figure 12). Surprisingly, all eight subjects with prostate cancer had detectable XMRV in the testes, even subjects for which XMRV was not detected in the prostate cancer tissues.

It's clear that we need to stop messing around with just the blood and get down to business with finding the virus where it lives. I'd literally give my left nut to move this thing forward (but let's hope it doesn't come to that).

 

[Cort]

It is frustrating basically knowing it is there! Hopefully Singhs autopsy paper will wake more people up. I can't imagine that it won't if it shows what we think it does....

 

[leela]

It's clear that we need to stop messing around with just the blood and get down to business with finding the virus where it lives. I'd literally give my left nut to move this thing forward (but let's hope it doesn't come to that).

For frack's sake! This has been bugging me since the beginning...why *does* everyone insist on looking in the blood when we know for scientific fact that is not where it is most prevalent?
But, erm, CBS, I sincerely wish all your body parts shall remain intact...

 

[Valb626]

CSB, thanks so much for the NCI info!! You "noted" that Coffin consults to NCI/DRP, right? (Save that man part, man!)

leela, in the intro slide to the BWG results, the presenter basically said they "really need" a reliable/valid blood test because that's most cost-effective for dealing with the blood supply, as well as for cheap, easy testing of individuals (us!).

 

[leela]

CSB, thanks so much for the NCI info!! You "noted" that Coffin consults to NCI/DRP, right? (Save that man part, man!)

leela, in the intro slide to the BWG results, the presenter basically said they "really need" a reliable/valid blood test because that's most cost-effective for dealing with the blood supply, as well as for cheap, easy testing of individuals (us!).

Okay, in this context, point taken. But in the continued debate about whether it exists at all, I think searching in tissues is essential.

 

[leaves]

Ok I probably am not getting most of it..could someone in a few sentencss tell me why Coffin is negative (as opposed to cautious) about xmrv, that is, how does that present itself?

 

[pictureofhealth]

Yes, I guess the BWG will be looking for a blood test above all. Not sure how this works though if Singh and others indicate that XMRV mainly lurks in tissue (after the initial acute infection).

However, I do remember Dr Singh suggesting that if the subjects (Macaque monkeys in this case) were injected with something to stimulate the immune system (there is a word for this I have forgotten!), then the virus re emerges from the tissues and starts re-circulating in the blood.

So maybe the way forward for the BWG is to stimulate the immune systems of the patient group and see if they have better/more consistent luck at turning up positive results for XMRV in greater numbers in blood after this stimulation/injection.

 

[omerbasket]

DHHS has expressed interest in the number of people who register for/attend the Association's webinar on the BWG study as a way of gauging the community's interest in the study. You can register here: https://www1.gotomeeting.com/register/985931313

Show them we're interested!

WTF?! So, for the DHHS it doesn't matter if people are sick, or if other people would get infected with a retrovirus that can cause ME/CFS, cancers and who knows what else - all they care about is how many people are interested? I mean, if just 10 people are interested - so they'll say "let the virus spread and let many more people get sick, without a good way to help them when they do"?
This seems to me very very disgraceful!

 

[urbantravels]

Take a breath, omerbasket. This is a positive message from the DHHS, not a negative. Not a "we'll cut off all funding and never get interested in this topic again if you patients don't show up" message, but a "we actually give a damn what the patient community thinks and whether they want more access to this information" message. THAT'S BEEN GREVIOUSLY ABSENT from health agency interactions with patients in the past decades and we should be jumping for joy every time we see indications that the health agencies' attitudes are changing.