Of shorts and blots

This article looks at specific accusations in the citizens petition supplement attachment 1

Example #1: “Manipulated WB”. The CP claims that different WBs were “spliced together” to make a figure in a 2005 paper. They point out a “bowtie” effect of several bands as supporting evidence. However, anyone with WB experience has seen this bowtie effect. In theory, it can be caused by proteins sticking to the sides of the wells or a small amount of extract entering the space between the wells and the plates that support the gel on each side. This effect is often seen when the top of the gel is exposed to air too long prior to loading.

Example #2: “Falsified WB”. This claim states that the same beta-actin control WB was used in 2 different papers published 5 years apart. The CP takes a beta-actin WB, which is low resolution, stretches it vertically, then blows it up and compares it to a WB published 5 years earlier. Although they claim the blots are identical, to me they look completely different. For example: 1) the left side of the band in lane 1 of the 2005 publication has a notch that is not in the 2010 WB; 2) band images between lanes 2 and 3 are clearly different; 3) the right sides of the bands in lane 4 are also different. I also have difficulty taking this claim seriously. The labs likely ran many beta-actin WBs between the 5-year timeframe the 2 papers were published, but it is claimed they somehow elected to risk their scientific careers to include 4 control lanes from a previous publication? A new beta-actin WB could be run in half a day. This accusation suggests that the scientists who contributed to the CP are not very experienced in molecular biology.

Example #3: “Reused WB”. This claim takes 2 WB panels of a figure and compares them side-by-side, suggesting they are identical. This claim could have some credibility, though it is more likely a mistake in constructing the figure for publication than scientific misconduct. Although I can’t say for sure that the 2 WBs in question are indeed the same, they do look very similar, though one is a slightly darker exposure. When WBs are exposed to film, we generally get several exposures from light to dark. If the films aren’t labelled properly, things can get mixed up when the graduate student/post-doc generates the figure for publication, especially if two of the WBs look similar. If the 2 blots are indeed the same, the PI, other authors, or reviewers probably should have asked if this was indeed an error. I’m inclined to think this was an error in making the figure and not intentional since there is nothing covert going on here- the 2 WBs are basically side-by-side in the same figure.

Example #4: “Band insertion into WBs”. The concerns raised here include: 1) irregular spacing between lanes; 2) FLNA bands not looking correct since it is a large (290 kDa) protein; 3) bands looking identical between lanes; and 4) white halos around bands. In my opinion, this claim is completely baseless and shows a lack of experience of the scientists that generated the CP. Irregular spacing between bands is routinely seen with self-poured gels. It is explained by the teeth of the comb used to cast the gel not being completely straight when the gel is polymerized. The claim that FLNA shouldn’t run very far in a gel because of its large size is inaccurate because proteins will run as far through a gel as a current is applied. Longer gels could have been used. Also, the researchers could have used gradient gels that allow for larger proteins to be better resolved in the gel. As far as the control beta-actin bands looking identical, they are basing this assumption on a common “tadpole-like” appearance of the bands. However, this tadpole effect is commonly seen if the gel is polymerized next to an air current (e.g. in a fume hood). The air current can cause the sides of the wells to lose moisture during polymerization resulting in a tadpole-like effect on the same sides of the wells of each lane, which is seen here. As far as halos around bands, most films used to expose WBs have a threshold level of exposure that is designed to limit background. You often see this halo effect directly on the exposed film, especially with stronger bands. The white halo could also be a compression effect, caused when the figure levels were adjusted for publication.

Concern 3.2 “Evidence of data manipulation from human tissue”. It is claimed that Figure 12 of a 2017 paper shows a WB with 12 lanes for the control protein NR1 and 13 lanes for PCLgamma1. It is also suggested that WBs from different experiments were spliced together to make the figure. The 12/13 lane issue is an obvious error that likely occurred when making the figure for publication, but in no way rises to the level of scientific misconduct. As far as splicing together 2 different WBs, please refer to Response to General Concern #1 above about manipulating low-resolution figures and spliced WBs. I honestly don’t see much evidence of splicing for the top WB. Also, on the bottom WB, the change in pixels appears to wrap around the top of the band on the left side of the proposed splice site. It would be difficult to crop the figure this way. Another thing to point out is that the 3 lanes on the right of both WBs are running lower in the gel than the band directly to the left, which is slightly higher than the other bands to its left. However, the proposed splice sites are 4 lanes from the right on the top WB and 3 lanes from the right on the bottom WB. These factors support that the figure was not generated by splicing 2 WBs spliced together.

Appendix items/Additional areas of concern:

Claim #4: It is claimed that the top WBs cannot be FLNA based on the banding pattern. Please see Response to Example #4 above. Also, note that Albumin was used as the control protein here, which is a comparatively large (66 kDa) for a control protein and suitable for analysis of large proteins, such as FLNA.

Claim #6: It is claimed that several papers between 2010-2017 don’t show control WBs for co-immunoprecipitation experiments that evaluate the interaction of beta-amyloid with alpha7-nicotinic acetylcholine receptors. This claim is true, but there is a very good reason for it. Beta-amyloid are small (10 kDa) peptides of 40-43 amino acids in length. Their small size makes them very difficult to analyze by WB, other than running specialized gels that would likely preclude the analysis of larger interacting proteins, such as alpha7nAChR (60+ kDa). Most companies pre-validate their antibodies for use in certain applications, such as immunoprecipitation experiments.

E.2 Additional suspicious WBs (1) and probable band duplication (2):

1) Suspicious WBs. The CP shows several examples (pages 30-31) of control bands looking the same between WBs suggesting that the researchers must have duplicated these images. To me, this is a ridiculous claim. I see clear differences between the bands in question and I think most others do as well.

2A) Probable band duplication. Regarding the white halo around bands on WBs, see Response to Example #4 above.

2B) FLNA bands look identical (page 33). The CP highlights 3 FLNA bands that they claim are duplicated, but I see clear differences. For example, differences are seen with the bottom of the right and middle bands and top of the right bands.

2C) Five IRbeta bands look identical (page 34). There are clear differences in the sizes of the notch at the bottom left of each band, as well as the slopes of the bands on the top right.

2D) Clipping multiple blots together (page 35). The clip effect that is seen when the proteins run through an air bubble in the gel, which is common with self-poured gels.

2E) Tampering of WBs (pages 37-39). It is claimed that bands are inserted into various WBs in a 2006 Nature Medicine publication. This claim is borderline ridiculous and is likely why it appears at the end of the CP. Dr. Wang is first author on this publication, so the implication being made is that he started manipulating data early in his career and continues to do so today as head of an academic laboratory. Firstly, Nature Medicine is an outstanding journal, so the data was thoroughly vetted by experts in the field prior to publication. See Response to General Concern #1 regarding manipulation of low-resolution WB images and drawing conclusions. Also, several of the examples shown as evidence of data manipulation support the opposite. For example, on page 38 the background pixels around the bands are irregularly shaped. It is hard to believe that Dr. Wang would have cropped each of the bands for insertion as irregular shapes (which would be difficult in 2006) instead of just using a box shape. Also, if differences in background pixels are evidence of data manipulation, why are there also irregular shaped pixels around the text in the figures? Were they copied from other figures as well? It is obvious that the differences in background pixels observed are the direct result of the scientists who constructed the CP altering the contrast and brightness of already low-resolution figures.

Notes from a Molecular Biologist

The author of this post received a Ph.D. degree in Molecular Biology and has been an academic researcher since 2003. His laboratory studies cancer and other human diseases and routinely run western blots (~1,000/year) for their studies.

I thought it might be helpful to post an expert’s review of the concerns raised in the Labaton Sucharow Citizen Petition (CP) regarding western blot (WB) data for Cassava Science’s Alzheimer’s drug. Simufilam has demonstrated remarkable improvement in cognition in Alzheimer’s patients at 12 months of treatment in an open label study and the drug is entering two pivotal phase III studies.

Western blotting is a research tool that we use to analyze proteins, including their levels, modifications, and association with other proteins. Protein extracts are prepared from cells or tissue, denatured using a detergent and heating, and then loaded onto a gel, which is a matrix of polyacrylamide, and then a current applied. This allows for proteins to be separated based on their size (i.e. molecular weight (MW)). After the gel is “run”, the proteins are transferred (i.e. blotted) onto a membrane and then probed with an antibody specific for whatever protein you want to analyze. The blot is then probed again, this time with a secondary antibody that is enzyme-linked and binds to the first antibody, thus allowing for detection using a chemiluminescent reagent that produces light where the first antibody binds the membrane (thus producing what is known as a “band”). All WBs include analysis of a control protein (e.g. actin, GAPDH) to confirm that equal amounts of protein extract were loaded in each lane.

The first thing to understand about WBs is that they are semi-quantitative. They can basically tell you if there are differences in levels (increases, decreases) or presence of a modification or binding protein. Secondly, the western blotting technique is somewhat of an artform. It takes practice knowing what procedures are best to analyze a specific protein. Thirdly, there are a lot of variables. Researchers use different first and second antibodies, gel conditions, membrane supports, washing and exposing conditions. There is no standardized protocol. For example, many academic labs that are on a budget will pour their own gels as opposed to buying more expensive pre-made gels. With the former, you have more control over your analysis, but the WBs can often look a little dirtier, lanes slightly unevenly spaced, or bands looking not so sharp. Though the results, in terms of levels etc., should be the same no matter what variations are used.

My overall impression of the CP is that it does exactly what it set out to do- confuse the average investor and seed doubt so that they sell their shares. The average investor has no idea what a WB is let alone how to objectively analyze them. The majority of concerns raised are easily explained and a small number of others are obvious errors that likely occurred during generating figures for publication. There is no obvious evidence of systematic data manipulation or scientific misconduct. Someone with knowledge of molecular biology likely helped put together the CP, but many of the concerns raised show a shallow understanding of WB technique and data analysis. Below, I provide responses to general concerns and then a point-by-point analysis of each concern raised in the CP.

General concerns raised in the petition:

1.    It is stated that “the western blot data presented by Wang/Burns are almost always overexposed and highly processed, which has been repeatedly seen in previously reported examples of image manipulation.” Several of the studies scrutinized in the CP were published between 2005-2012. It is important to realize that during this time WBs were visualized by exposing the membrane to film. Control proteins generally give very strong signals due to their high level of expression in cells and use of validated antibodies that give the best (i.e. strongest) signal. Also, analysis of low-expressed proteins requires more extract to be run per lane to see the signal. These factors can often lead to control bands that are strong (i.e. overexposed). Today, most researchers use digital imaging systems to expose WBs, which avoids issues with overexposure. As far as the claim of “highly processed” images, know that most research papers published pre-2010 used images of 300-600 dpi or lower. Images retrieved from online papers, as was done for the CP, are likely even lower resolution. In my opinion, it is dangerous to take a low-resolution WB and adjust the contrast and brightness levels to attempt to uncover evidence of data manipulation. For example, the CP repeatedly shows changes in background pixels as evidence of data manipulation, but there are many factors that could give this effect. Over the years, I’ve seen things as simple as streaks on a film caused by the processor or how the membrane is wrapped with layers of plastic wrap to prevent drying from influencing the background of the figure.

2.    It is repeatedly said in the CP that the authors should produce the original WB data in order to satisfy their concerns. I’m pretty sure the scientists behind the CP know that this request is unreasonable. Most researchers will store data after publication in case questions do arise. In a perfect world, films and electronic WB data would be stored forever, but students leave the lab, labs downsize or move to new institutes, and thumb drives get lost. In addition, journals generally don’t require researchers to keep data for longer than a year or so. The fact that Drs. Wang/Burns have not “answered” the WB concerns of the CP is likely not due to them hiding something but rather that it is impossible to retrieve data used in a publication 10-15 years ago. Journals rely on a thorough analysis of the data by the reviewers, which are generally 3 experts in the field. If a reviewer had a problem with any of the WBs in question, they could have asked for them to be repeated. If they thought the data were manipulated in any way, the reviewers would have instantly rejected the paper and likely notified the journal.

3.    The CP repeatedly says that their analysis indicates fraud or scientific misconduct. Scientific misconduct is a very serious claim. As scientists, we welcome critiques of our data presented in research papers or presentations. That is part of being a scientist. But know that scientists are trained from early in their careers and throughout about what constitutes misconduct. We take refresher courses and sign statements when we submit research papers regarding the validity of our work. Every researcher knows that being accused of misconduct can instantly ruin a career. In my years as an academic researcher, I have never known of anyone who was even accused of scientific misconduct. I’m not saying that it doesn’t happen, but there is a big difference between a paper mill in China that is duplicating figures in different publications and an established researcher whose lab has consistently published quality papers and is supported by NIH funding. It is improbable to think that Drs. Wang/Burns have systematically produced fraudulent research and manipulated data for research publications and grant applications without a colleague, collaborator, or reviewer detecting it for 15+ years.

In the next article, I will follow up on specific allegations made in the petition.

Citizen Petition Supplement

The petition against Cassava Sciences was supplemented on Sep 8. We shall examine some questions arising from this supplement and provide our comments in this post.

Concern #1: Authenticity and Availability of Phase 2a Data—Western Blots

The petition claims the original western blot images should be available at Cassava’s premises
Can someone point us to a regulation that requires this?

The petition claims corresponding authors in a paper must store, maintain, and validate data.
The role of the corresponding author according to International Committee of Medical Journal Editors (ICMJE) is shown below

Source :http://www.icmje.org/recommendations/browse/roles-and-responsibilities/defining-the-role-of-authors-and-contributors.html

Concern #2: Phase 2b Biomarker Redo Was Not Done by an “Outside Lab”
Concern #3: Which Outside Lab Did the First Phase 2b Biomarker Study?

These concerns were already presented in the original petition and are just repeated in the supplement. Here is the relevant paragraph from Cassava Sciences’ Form 10-K where they mention that blinded conditions were maintained.

Source: https://www.cassavasciences.com/static-files/0e33310f-8cec-4c7b-8444-8ae4bc25116f

We also tried to find other study protocol designs where names of the specific lab(s) were listed and mostly came away empty-handed. Most big and small pharmas do not list the names of the labs they use; many do not even upload study protocols or results onto clinicaltrials.gov. This list includes drugs that were granted SPA. When we did find a trial granted SPA with a study protocol, the shipping address for the samples in that study was blacked out. But by carefully studying the protocol we found that the shipping recipient was the sponsoring pharma. Our point here is simple – the independence of labs is only truly guaranteed by blinding requirements and not affiliation. If this were not the case, it would be impossible to trust trial sites and labs used in developing countries, as many big pharmas do.

On this front we plan to watch out for the study design of Cassava Sciences’ phase 3 studies for Simufilam where we may learn how they plan to leverage their new CRO.

Concern #4 Likely Lab Errors or Manipulation in the Phase 2b Redo Study

The petition claims that CSF/plasma albumin ratio baseline does not match clinically observed ratios

In the methods section of the phase 2b paper, it is mentioned that optical densitometry is used in determining CSF albumin levels. It is therefore fairly clear that the CSF/Plasma albumin baseline ratio also used the same measure. Our team member highlighted this fact on twitter first. 

Albumin constitutes a high portion of plasma protein (~60%) and can easily hit the upper end of the optical density range due to saturation. This usually causes an under measurement of plasma albumin and can bump up the CSF/plasma albumin ratio significantly as seen in the baseline provided by Cassava. The clinical ratio Qalb, which is usually measured by a more precise ELISA assay, cannot therefore be directly compared to this technique.

In Cassavas’ defense, the change in CSF albumin (not the ratio) is the biomarker of interest and the comparisons there between placebo and dosage arms show a treatment effect and a positive reduction of CSF albumin.

We also want to point out the malice in the chart included in the supplement as seen below.

The petition referred to multiple past studies where the CSF/Albumin ratio was reported in the range 4 -9. This is, in reality, 0.004 – 0.009 (without the 1000x factor to make QAlb read more easily in clinical charts). They placed Cassava’s value of ~0.25 alongside this in an attempt to magnify the difference. This is extremely misleading and can easily be shared on social media to propagate misinformation. The supplement conveniently omits mentioning the use of optical density in the paper. As a result, the above misleading chart is completely lacking context.

The petition claims that CSF IL-6 baseline does not match clinically observed ratios

The petitioner states that “In other AD studies, CSF IL-6 levels are 1-5 pg/mg.” That’s actually a typo and should be “pg/ml.” But let’s assume they meant 1-5 pg/ml. They further state that the baseline IL-6 levels for Cassavas’ studies are 33-34 pg/ml and are far outside the norm (they volunteered to provide references on request). Why are the values so different? It seems as if all the “Exemplary published studies” referenced in the petitioner’s supplement used ECLIA (Electro-chemiluminescence Immunoassay) in the measurement of CSF IL-6 levels whereas Wang et al used ELISA (Enzyme Linked Immunosorbent Assay). It was even stated in Dr. Wang’s publication, “Although these assays are not directly comparable, values from each are reported in pg/mL.” In essence, they are correct in pointing out that the values are drastically different. But what they fail to point out, however, is that they were measured using different methods. It is a continuation of the same theme of misleading comparisons made with seemingly incriminating charts.

Gruber et al., showed elevated CSF IL-6 levels for a variety of conditions. These levels were measured using ELISA. [2][3]

·   Neuro-lupus (71.40±5.89pg/mL)

·   CNS infection (374.24±92.61 pg/mL)

·   Other inflammatory, neurological conditions (33.92±29.36 pg/mL)

·   Controls without inflammatory CNS disease (4.35±3.00 pg/mL)

Note that the CSF IL-6 baseline values measured by Dr. Wang et al fit well within the inflammatory, neurological conditions range above. Again – both methods being ELISA.

We acknowledge that ECLIA may be a superior method over ELISA, but “Currently, enzyme-linked immunoassay (ELISA) is the most widely used approach for the detection of AD core biomarkers in CSF. However, these ELISA methods often show considerable inter and intra-lab variability that prevents the use of standard cut-off values and precludes the wide use of CSF biomarkers in clinical practice.” (reference). Regardless of the CSF IL-6 baseline values, or how they were obtained, it is crucial to note that it is the change from baseline that is important. And that is what was measured in the Phase 2b study.

The petition claims that CSF sTREM2 baseline does not match clinically observed levels

Similarly, the petitioner claims that the sTREM2 levels obtained by Cassava Sciences are far lower than other published studies. Another simple literature search can find studies with measured CSF sTREM2 levels even lower than Cassava’s, in the range of 172.5 to 305.4 pg/ml. (Heslegrave et al. Molecular Neurodegeneration (2016) 11:3)

Concern #5 Apparent False and/or Misstatements in Cassava’s Phase 3 SPA Request

We did not find any deliberate misstatements. There were some errors, but minor errors do not constitute fraud and do not in any way call into question the safety of Simufilam or its potential efficacy. In fact, publishing all this data and facing scrutiny is what has set up Cassava for a more robust rollout of its phase 3 trials. 

Authors conclusion

Remi Barbier’s recent fireside chat clarified a few things. Phase 3 trials are on track, and he confirmed that cognition and biomarker improvements are not tainted and show real treatment effects. He stated that while the results are encouraging, clear links have not been established between cognition and biomarkers. We appreciate him for volunteering to provide transparency on a chain of transfer of data relating to the upcoming 12-month results of the Open Label study. This shows confidence and will serve to reassure investors. We eagerly await those 12-month results. In a recently released corporate presentation, it was specified that there is a < 10% drop rate for patients transitioning to the CMS study – this is a very low rate that shows patients are willing to risk being put on placebo. 

In summary, we find the fraud allegations in the citizen petition are filled with malice as they continue to attempt to discredit Simufilam and Cassava. The allegations do not explain how phase 2 clinical results show improvements in cognition scores measured across multiple clinical trial sites and are obfuscating the blinding processes put in place. 

We are confident that the FDA will reject the allegations in the petition.

Note: In the event of any verifiable errors, we will take steps to update the post. Please add comments on the blog to help us ensure the accuracy of our findings. Thank you!

Science Integrity – Part 1

Dr. Elizabeth Bik is a renowned science consultant who runs a blog and a twitter account where she discusses manipulated images in scientific publications. Her work is widely respected in the scientific community. Her twitter bio reads

Scientific discussions should not be held in the courtroom

In this post we will examine her recent actions with respect to Cassava Sciences. Below are some of her first posts on the subject;

Source: twitter

Dr. Bik was quick to validate the petition, in particular the fraud claims pertaining to inconsistent western blots. She later posted her comments on pubpeer while giving the authors a chance to make any corrections. The contributors of this blog article agree with her concerns on these topics.

Next she followed it up with a blog post.

Source: twitter

The papers in question are at least a decade old and within 2 business days of posting the initial tweet, she was already talking dollars involved and tagging cassava investors with her unproven allegations. To be fair, her tweet above was in response to Cassavas initial fact/fiction response which was dismissive of the petition. Even so, one would expect Dr. Bik to hear back from the scientist(s) in question instead of using company PR as a reason to insinuate fraud against scientists and scare investors. Note that Dr. Bik has this disclosure on her blog page.

Source: https://scienceintegritydigest.com/2021/08/27/cassava-sciences-of-stocks-and-blots/

Question to Dr. Bik: What was the motive behind that tweet?

Later we were dismayed by Dr. Bik not following up on many requests to examine the high resolution western blots on the patent by Dr. Wang. She also chose not to engage or update her blog when she received responses like the ones below – instead choosing to highlight the threats from abusive trolls on twitter.

Source: twitter

Her next twitter post was a new blog post that highlighted another of the original petition’s charges on Cassava Sciences which was the poster presentation at AAIC. Meanwhile, that ticker symbol she is highlighting everyday in her tweets, is taking a hit causing shorts to pile on and heavy losses for knowledgeable investors. Note that we think her work on the blog article about the poster was unprofessional and didn’t meet the high standards she is renown for. We will be commenting on her work on the poster and plasma ptau in upcoming posts.

Source: twitter

Overall it was obvious that a scientific discussion was being held in Dr. Bik’s courtroom where she seemed to be judge, jury and executioner. First she piled on with short interests questioning a company she barely knew much about. Next she had no problem with her work being used to supplement the petition that questioned the integrity of the Cassava team and Dr. Wang. And finally while ignoring Dr. Burns, she also complained about how women of science like her were being downplayed by techbros.

We have more questions for Dr. Bik.

  • Can Dr. Bik say with conviction that she is a neutral arbiter and not an interventionist supporting the fraud claims?
  • Can she declare that her associations with Adam Feurestein, Prof. Rob Howard Prof. Lon Schneider and other critics did not cloud her view of Cassava Sciences?
  • Does she support the intent of the petition asking for halting of drug trials based on unproven allegations from some of her work?
  • Can she call out Adam Feurestein for his stat news article which engaged in veiled sexism and questioned the credentials of an accomplished woman scientist?
Authors conclusion

The authors of this blog acknowledge Dr. Bik’s renowned expertise in spotting errors/issues and falsifications in academic journal publications. However with her actions surrounding the allegations on Cassava Sciences, we question her motives and invite her to engage in a balanced scientific discussion with us the authors of this blog on any concerns regarding the company. We also challenge her to hold herself to the high standards she has set.

The Hyper Growth Story

Any statements on hyper growth must be prefaced by answers to the accusations on the petition

Presence of a Whistleblower

This one is obvious – there wasn’t any. The petitioner used the term once and that too in the title of the petition. It was merely a ploy to garner increased attention. They being short on the stock, this smells of manipulation and we hope the SEC investigates them for inducing hysteria.

Dr. Wang’s papers and experiments (western blots, tissue images, experiment concerns)

We don’t know the truth here and don’t want to speculate. As Dr. Bik suggests, the original high resolution images could help alleviate any concerns and if there were errors, Dr. Wang may be able to correct them.

Our concern with the experiments being fudged are low because we were able to find complementary research by a team at Yale as described in this paper. Here they were able to determine that targeting elevated levels of aberrant Filamin A with PTI-125 reduced seizure activity by over 60% in mice when compared to saline. They ran several experiments like treating neonatal mice with a genome type that cause seizures with PTI-125 and also treating mice that were already experiencing seizures for many weeks. Results were extremely positive in all their experiments.

One can also read about research on Filamin A and diseases caused by mutations of the protein

Joe Springer, a Seeking alpha contributor also listed a host of complementary research here. Here in this blog, we are working on an article that will attempt to explain the MoA of the drug. Please subscribe for an update.

SavaDx poster

After corrections to the poster data, measurements done with the plasma assay showed a significant correlation between Simufilam use and p-tau181 decline. This matches the strong correlation that was also seen in the OLE study between cognition and p-tau181 decline.

The number of patients being < 64 is not a concern as they clearly marked the criteria for p-tau181 evaluation in the posters methods section. We also posted earlier on why the poster data is not relevant to phase 3 studies.

Independence of the CUNY lab

This particular point is prone to the most speculation. Here are some details on the study.

The masking requirements are clearly specified. Those concerned with the rigor of following these guidelines can read documents from the FDA here and here. Also the following excerpt gives more information.

Source: https://www.clinicalleader.com/doc/data-integrity-in-clinical-trials-key-concerns-of-the-fda-mhra-0001

Cassava has also confirmed that they use independent bio-statisticians to analyze the data obtained from primary and secondary outcomes. In the phase 2b study they used axiom real time metrics.

Cassava also released a press statement after the reanalysis of phase2b samples. It can be observed below that the blinding was maintained even though there is some confusion on the difference between outside labs and the academic lab. It would be nice if Cassava clarifies this.

Source: cassavasciences.com/news-releases
Concluding remarks on the petition

Our view is, the FDA verified all processes were followed correctly before granting SPA for phase 3 trials. We also think the petition will be dismissed in due course because of the obvious conflict of interest of the petitioners and the fact that safety concerns were never even raised (the primary purpose of a citizen petition).

One additional point to note is that in comparison with many other AD trials listed on clinicaltrials.gov, Cassava have been one of the few companies that has been diligent in updating the database with protocol data and results. They have invited the scientific community and investors to scrutinize those results. One must also note that many big bio pharmas tend to use their own affiliated labs and don’t even list them publicly.

Here is CEO Remi Barbier’s statement on the phase 3 trials,

Source: cassavasciences.com

Criticisms of the Open Label trial

Dr. Burns and her team of scientists have persistently reminded us that the OLE trial does not prove Simufilam is effective without confirmatory phase 3 studies and there will be risks. However the smart investor can see some positive signals from these trials. As discussed previously, the impressive cognition results at 9 months is in comparison with a placebo baseline from a meta analysis of over 20,000 patients with mild to moderate AD. Also it has been researched that the placebo effect for AD patients begins to decline after ~6 months. Even if placebo was in effect, showing improvements in cognition for 9 months, where decline is the norm is truly ground breaking. If we see these results repeat for the next 50 patients and at 12 months, one should really start to feel good even without the results of the p3 trials. Cassava have also initiated a double blind Cognition Maintenance Study with randomized placebo control that proceeds to extend the duration of patients getting treated with Simufilam in the OLE trial and we hope to see interim results from that study the first half of next year.

The Hyper-growth story

For a company that is being accused of misconduct and false claims, Cassava sure isn’t playing it slow and milking investor cash. Their cash spend is low, insiders haven’t sold, even bonus targets that were met weren’t awarded. All this to preserve cash for all the expensive trials they are running. They are also working hard and fast with the FDA to run multiple studies to collect as much data as possible for approval.

We fully believe Cassavas growth story can continue and don’t think anything has significantly changed except for the stock price becoming cheaper. We are buyers at these levels. Here are some of our predictions,

  • Continued cognition improvements in a majority of the 50 patients in the OLE trial at 12 months with data being released by early October
  • Phase3 trials begin recruiting mid september with large interest driven by word of mouth
  • A potential partnership deal that may be delayed because of all the bad press
  • Announcement of trials for new indications
  • Data release from more patients on the current OLE trial.
  • Break through therapy designation

Reason for caution

Our chief concern at this point is the extra scrutiny around the company and if there were any concerns from the FDA on how the blinding was maintained during pandemic times. We don’t have access to the end of phase 2 meeting minutes. However if Dr. Wang’s academic lab maintained blinding standards and there were no concerns raised by the FDA in those minutes then the concerns will shift to successful outcomes in the phase 3 trial.

In conclusion

We hope this article will switch you from the fear and uncertainty being propagated by short interests and instead look at the Cassava team in a new light. We hope the Cassava team are more diligent about how they present results and hope they use the extra scrutiny to make less errors while continuing to be transparent

Good luck to all!

AAIC Poster and Phase 3

The poster presentation at AAIC had some errors and this was exploited by the petition. We will discuss the errors at length on another post.

This post however is to inform readers that SavaDx or plasma p-tau end points were not part of the phase 2b trial and are not subject to the blinding requirements. Cassava independently chose to get their SavaDx test assay verified by correlating it with p-tau results from the commercial assay run by Quanterix. This was purely to show SavaDx as a valid biomarker while also showing how reduced altered FLNA helped with lowering p-tau181.

Any errors on the poster therefore should not impact phase 3 decisions by the FDA.

Dr. Burns at AAIC ’21

Alzheimer’s Association International conference (AAIC) was held on July 29th – Cassava Sciences presented 9 month cognition data for the first 50 patients enrolled in their open label study. Their slide deck was presented word for word by Dr. Lindsay Burns, SVP and Lead Scientist.

The company used the term encouraging and made no claims of efficacy while describing their results. However, that did not stop many from weighing in. Here are some social media criticisms posted by well known influencers –

Source: twitter

An excerpt from the Adam Feuerstein’s article above:

It’s not hard to see past Adam Feuerstein’s implied misogyny and lack of basic research on a publicly available study design. For the record, Dr. Burns, an author of multiple scientific papers, has the following background

Source: cassavasciences.com/management

Facing criticism for his remarks, Dr. Rob Howard, one of the quoted scientists on the stat+ article, doubled down and posted on his twitter feed.

Source: twitter

Dr. Howard ignored the questions surrounding his role in legitimizing the article while only responding to comments from fans and the harassment from reddit trolls. As for comments from users who asked him to take a look at the data and research – crickets.

Their criticism doesn’t change facts. Dr. Burns emphasized this was an open label study and only large phase 3 studies can prove efficacy. How is doing this overblown, uninterpretable or naive? Is being invited to present inappropriate? Before delivering the presentation, cassava had also announced two phase 3 studies.

It’s hard not to question the motives of these personalities and their backers when they engage in rhetoric. Note that their comments were picked up by various media articles to enable the “sell the news” effect that caused the stock to crater ~50%.

Some questions for these social media influencers,

  • Are companies that complete open label studies not allowed to present results to academia and investors?
  • Is your platform intended to foster progress with constructive criticism or plainly serve as a signal to short sellers and day traders?
  • Are you willing to be gracious and admit missteps when wrong?

There exists a world of folks with honorable reputations, who make questionable comments and never leave their echo chambers. They are mostly right but will never admit it when wrong. This blog hopes to critique these positions.

Author’s thesis on 9 month cognition release

The author of this piece acknowledges that Simufilam is not a proven drug. Preclinical studies on mice models and post mortem human brains have illustrated the mechanism of action. Phase 2 trials have shown encouraging results. The ongoing open label study is using a placebo baseline from a meta-analysis of over 20,000 patients with mild to moderate AD. Since a clinical hallmark of Alzheimer’s disease is decline in cognitive function, the improvements shown over 9 months, despite the placebo effect, is unheard of. The small sample size is the biggest concern and any investment should factor that in.

Biomarker results backing cognition were impressive. Here again it must be noted that biomarkers for Alzheimer’s disease is an active subject of research and the correlation between biomarkers and disease progression hasn’t been completely established. Cassava in their studies so far have found decreases in CSF p-tau 181 best correlates with cognition improvement.

Introduction & Cassava Sciences

This blog was setup to promote small cap bios in the Alzheimers and CNS spaces. Many small biotechs are forced to battle misinformation from social media scientists, wall street short sellers and pump & dump traders. This leads to attacks on the credibility of founders and scientists and causes extreme volatility in share prices.

In such scenarios, some small retail investors who have done their due diligence and who may have critical information to share, often struggle to disseminate information that counters the narrative. For instance these investors may tweet at a widely followed account of a social media scientist or write a comment on the authors blog and receive no responses. Sometimes the errors propagated may be so egregious and even acknowledged by the social media figure but then no follow up actions or corrections are made.

What this blog will specifically do for investors?

  • Provide a platform for vetted retail investors to publish scientific views that counter the prevailing social media sentiment on small cap bios
  • Research the science and provide general insights/opinions including critque of the critics
  • Provide a Q&A with experts in the field

What this blog will not do?

  • Be a platform for unscientific views or promote any sort of hate or misogyny
  • Receive any sort of in kind donation from investors or companies and people associated

The author(s) on this blog may hold short/long positions on the stocks.

The first company we will be covering is Cassava Sciences (Ticker: SAVA), a small cap bio, with its lead drug candidate Simufilam and plasma biomarker candidate SavaDx. A recent FDA petition filed by law firm Labaton Sucharow called into question the science behind the company and asked to halt its phase 3 trials. We will be examining the truth to the allegations using crowdsourced knowledge and responses from Cassava sciences (here and more recently here).

We hope you are open to our views!