Amarantus Bioscience Holdings Inc (AMBS)

[joboggi]

From the recent WSJ article. Note that they mention prevention of alzheimer's and the test that currently follows Ad

http://online.wsj.com/articles/alzheimers-disease-fight-focuses-on-preventive-treatment-1405396803

“Also, a greater ability to measure the progression of the disease in the brain through the use of biological markers, such as the imaging of disease-related proteins, has made it easier to detect the subtle and slow progression of the disease in live humans. Before these biomarker tests, Alzheimer's was diagnosed solely based on clinical symptoms. (It often still is.) Its pathology in the brain could only be examined with autopsy.”

There is only ONE test that can measure the progression of the disease. That is Amyvid. Note that AMyvid is said to detect the sublte and slow progression of the disease in live humans. There is only one test that does this. Amyvid. There will probably only ever be one test that does this. Lympro has not been shown to do this, and it is not set up to monitor progression.
___________________________

Poster #1.
http://content.stockpr.com/amarantus/files/pdf/Poster+1+FINAL+FINAL.pdf
First paragraph
“
• Background: Initial studies by Thomas Arendt et.al, at the University of Leipzig identified the
mitogenic response of peripheral blood lymphocytes (PLB) as a potential biomarker of Alzheimer’s
disease (AD) [Stieler, JT, and Arendt, T.; et al. Neuroreport 2001; 12(18):3969-3972]. This finding
was confirmed in a second study conducted by Jens Steiler, et al, where the authors were able to
retrospectively differentiate AD from Other Dementias [(OD), mostly Idiopathic Parkinson’s disease]
with 95% sensitivity and 90% specificity [Steiler, J et al, 2012. Neurobio Aging 33:234-341].”

Well, there are two mistakes here. Firstly, ALL of the other dementias were PD dementias. Secondly, if you look up that reference, it was 92% sen and 91% spec.

http://www-haproxy-e-c01.aws.barchart.com/plmodules/?module=secFilings&filingid=9500339&type=CONVPDF&popup=1&override=1&symbol=AMBS

This is very sloppy work.

Going on through poster number one.

“Methods: Analytical performance of this flow cytometer assay was re-established at a contract
GLP laboratory. In collaboration with clinicians, patient blood was sampled and purified for PBMCs.
Following the published protocol, a stimulation index was established for CD69 positive expression
following mitogenic stimulation. Experiments were conducted on 12 healthy volunteer donor blood
specimens to assess analytical performance characteristics of the assay. Pre-analytical and
analytical variance was assessed in healthy normal subjects.
• Results: Analytical performance data shows excellent reproducibility with coefficients of variation
less than 20% for most mitogenic conditions. Additional performance metrics like effect of gating
parameters and reagent stability were quantified. The effect of pre-analytical variables such as
sample handing conditions were determined as well.
• Conclusion: The LymPro test, which measures mitogenic response to peripheral blood lymphocytes, demonstrates suitable analytical performance for use in a fit-for-purpose fashion in the
Company’s hands”
___________________________
So, this poster was a validation study of the Lympro test. Keeping in mind that this statement does not take away from the study, it just defines it, this study does not include anyone with AD, or early aD or any dementia. It was not meant to, this poster talks about a validation study for the test itself in 12 healthy volunteers. The test showed excellent reproducibility which is fine. You cannot say anything about the test in AD based on this study. It was not meant to do that.

Then the poster goes over data about how the body works in AD. This is not related to the testing since no one in this study had AD or MCI.

Then table four and five go over the actual results of the test, which are then summarized

Summary

The Total CD69+ (as a % of CD45+ Lymphocytes) displays:
• Consistently low CVs in general and across stimulation conditions
• No effect of PBMC harvest point (Fresh or 24 hr)
• No inter-individual effect on CV in GLM models
• No inter-individual effect on Mean value in GLM models (which may indicate a tight
distribution in the healthy control population)
The Total CD69+ (as a % of CD19+) also displays consistently low CVs as well as
lack of significant difference on CV by stimulation condition, but shows an effect of
harvest point and an inter-individual effect on CV”
_______________________
These things basically say that the test is stable and usable.
However the conclusion has nothing to do with any of this.
There is no basis in this statement. “The test will transition into multiple clinical studies for differentiation of individuals at risk for Alzheimer’s dementia from other dementias.

No self respecting scientist would allow him or herself to conclude this based on the data. Not one.

Going back to the link JUST below the Wall Street Journal link, you will see that Lympro was only studied in AD and PD dementia. There are around 10 dementias, the most important of which are MCI and mixed, vascular dementia, and Dementia with Lewy bodies and several more. For some reason, the Lympro test is going to be used to differentiate all of these dementias from AD even though its never been studied for these entities.

Moreover, if you look at the lympro score for the Pd dementia patients, strangely their score is lower than the score for healthy subjects. This difference translates into the difference in Sen and spec between AD and PD dementia and Ad and healthy non ad people. For that number see the reference in the stickie on Lympro.

____________________________________________

http://content.stockpr.com/amarantus/files/pdf/Poster+2+-+Clinical+FINAL_PRINTER.pdf

Poster number two

Abstract
“ A blood based test for Alzheimer’s disease would be advantageous for early identification of
Alzheimer’s disease (AD). Multiple lines of evidence have identified Cell Cycle Dysfunction (CCD)
as a key pathology in Alzheimer’s disease. Furthermore, it appears likely that this dysfunction is
systemic, affecting Peripheral Blood Lymphocytes (PBLs) as well as neurons. Here we
summarize the clinical data of the Lymphocyte Proliferation (LymPro®) test as a means of
measuring CCD. The original findings [1] have been replicated and extended [2] with the
technique subsequently enhanced (“Version 2”) in unpublished pilot research presented here. The
LymPro test consists of measuring cell surface expression of CD69 in subsets of mitogenically
stimulated PBLs. Enhanced assay methods demonstrated up to a 2.5 fold increase in expression
of CD69 on multiple cell types. Using the enhanced assay methods in a small pilot trial and
employing multiple regression techniques, up to a 91% positive and 92% negative agreement
with subject clinical diagnosis. With further development, LymPro may become a useful blood
based biomarker.”

So far, I am not sure what is going on, because they are using the old numbers, 92% sen 91% spec, between AD and non AD dementia, which as you recall is actually PD Dementia, and not any of the many other dementias.

Methods Clinical subjects:
The Stieler 2001 study [1]: n=72; 27 with probable AD and 45 age-matched cognitively intact
(CI) controls.
The Stieler 2012 study [2]: n= 88; 32 with probable AD, 26 with Parkinson’s disease dementia
(PDD) (active control) and 30 age matched cognitively intact controls.
Enhanced assay study: n= 44; 15 with probable AD, 18 with other dementia (OD) and 11
cognitively intact controls.

We see that in the Stieler studies only PD dementia was studied. We are told that there are a small number of OD or other dementia patients studied in this poster presentation. That would be too small a number to be able to differentiate between AD and non AD dementias of which there are almost as many diseases as there are patients in this arm of the study. Each disease could have a separate profile, and probably does. ( Or not, we would never know unless it is studied specifically )

They go on to re report steiler one and two and then on to the new patients.

The new patients are presented in general terms. And the conclusions for the new patients are as follows. When you look at poster three, and at the footnote on poster two , you realize that the Enhanced Assay patients date to 2007, 2008. Really.

“All unpublished data presented on this poster was generated by Provista Life Sciences and prior to Amarantus acquiring the assets of Provista Life Sciences”

This covers the Enhanced Assay patients. When you read poster three you will see that these “enhanced” patients are the same patients as in poster three. You can check the numbers yourself.

“Enhanced Assay
•? Increasing concentration of mitogenic stimulants and longer incubation times
significantly increase the cell cycle activation marker CD69.
•? Increased CD69 expression appears to increase univariate differentiation.
•? Results demonstrate that multiple parameters can be tuned to optimize assay
performance, signal to noise, and maximize differentiation between Alzheimer's
disease and other chronic progressive dementia that confound diagnosis. “

So, one would hope that the new test translates into a broader difference between ad and non ad dementias, but they are not testing enough non ad dementias to make an assessment on that. Not by a long shot. There are almost as many types of dementia as there are people in the non ad dementia arm.

They also need to explain and test why PD dementia patients have lower lympro scores than healthy controls.
_____________________________________________

Poster number three.

http://content.stockpr.com/amarantus/files/pdf/Poster+3+-+Chart+Review+FINAL-APPROVED.pdf

Firstly, they DID change this from a retrospective study title to the proper title of a longitudinal study, which is correct. I guess they got an email from someone about what I wrote, or they read the boards.

Keeping in mind that they are talking about the enhanced assay patients from poster two.

Three people got excluded. Four people’s status were changed. That is almost too many and probably too many because of the total number of patients in the beginning which was 44.

You can read the reasons for exclusion and change.

They go on to say that “several” ad patients were found to also have Vascular dementia. Then they erroneously assume that those with Vascular dementia do not have changes in cell cycle dysfunction ( CCD, which would change their lympro score) They do not mention just how many Ad patients had VA added to their diagnosis. Va dementia patients have not been studied with lympro, so they cannot make that assumption.

Keep in mind that the numbers on the 44 patients were not previously published, so says poster number two. They exclude three patients, and then.

Cutting to the chase, when you look at the standard box that helps you set up the math for Sen, Spec, and accuracy, you see that the corrected results, based on longitudinal observation of the patients involved drops the Sen to 73% and the spec to 81% and the accuracy to 78%.
I did not make that up, it is right there on the chart under results for this study. You can read the reasons why people were changed and or left out.


Now these numbers also mean that any BD study on the current patients, even though it is too small in number also cannot be evaluated properly until the people go to autopsy. After following the patients, what you thought you had is not what you are going to wind up with, which is not unexpected.

So, after publishing data that shows that Enahnced Lympro is getting worse numbers in the longitudinal study, they go on to talk about what we all expected out of these posters.

What follows is a quote from the poster. It ends with the concussion summit internet address.

Ongoing work (as of July 15th, 2014) includes:
1) Bridging trial: A72 subject study is underway at Becton Dickinson employing the original and
enhanced assay stimulation parameters. Objectives include
a) Replicate the original data sets of Stieler et al, 2001 and Stieler et al, 2012
b) Further explore the enhanced stimulation techniques presented here to maximize differentiation
in stable assay environment at Amarantus’ contract laboratory, Becton Dickinson Biosciences,
Inc. (BD Biosciences, www.bd.com).
2) Top line results are expected to be presented on July 31, 2014 at the #C4CT Concussion
Awareness Conference at the United Nations.
3) Analytical performance:Analyte Performance/Validation Package suitable to support
pharmaceutical clinical trials will be completed in second half of 2014
4) Continued development:Amarantus intends to conduct multiple robust clinical performance
trials incorporating biomarker qualified AD and cognitively intact subjects, as well as multiple types
of other dementias, clinically validate LymPro.
5) Collaboration:Plans to work with academia and industry to support research activities.
Summary
Because a clinical diagnosis of dementia is not uncommonly made in error, we sought to take the
data from a 2008 pilot study of LymPro and increase the accuracy of the clinical diagnosis by
undertaking a chart review to attempt to clarify the diagnosis in 2014.
• The chart review clarified 7 original cohort designations, resulting in excluding three and a
change in cohort of 4, a change in 16%
• The increased precision of diagnosis yielded univariate accuracy less than 80% in select
subpopulations of lymphocytes
• ROC Area Under the Curve (AUC) range around 80% with multivariate models
• Sufficient evidence exists to support the further evaluation of LymPro across original and
optimized assay conditions
• An ongoing Bridging study will yield initial specificity and sensitity data for LymPro in original
and optimized assay conditions that will be presented at the #C4CT Concussion Awareness
Summit at the United Nations on July 31st, 2014 (www.c4ctsummit.com)
_____________________________

Comments on the above copied material from the poster.

1. 72 patients is not nearly enough to differentiate between Ad v non ad dementias, there are too many dementias to cover.

2. They make an assumption that Vascular dementia does not change the Lympro score. Are they going to make that with all non ad dementias, without testing them? They are assuming that the data from PD dementia carries over to all of the other dementias, and well, you never know until you test. I would expect that especially mixed dementia is part AD, and may well show a positive lympro score. Certainly, no researcher would assume that and use the result one way or the other to identify Ad when it has not been properly testing on all non ad dementias.

3. The published numbers do not improve on Amyvid, which is currently the standard.

4. The Lympro test is not tested to show improvement or worsening with worsening of symptoms as is shown with the Amyvid test. This is extremely important for researchers given the current thinking that worsening of the amyloid plaque corresponds with worsening disease.

5. I’ve never seen three posters that are this disjointed in their presentation.

6. They bury the updated sensitivity and specificity and accuracy data in a table and do not put it in their summary, which any good researcher would.

Finally, it looks like this cell cycle theory goes all the way back to 1991 based on the references for poster number three.
___________________________end poster review


http://www.alz.org/dementia/types-of-dementia.asp

http://www.caring.com/articles/types-of-dementia

http://my.clevelandclinic.org/disorders/dementia/hic_types_of_dementia.aspx

Here are three references about the different types of dementias. Lympro to date has been tested in AD and PD. There are many more.

I would put this in my stickie, but I will leave it here so that you do not have to wade through that. It is getting pretty long.

I am SURE that the folks at AMBS did not think that anyone who could actually read these posters would go over them. This is farcical.