Sample Cancer Test Misinformation

Source: http://rationalwiki.org/wiki/RGCC_test

After I received a forwarded link of the Cancer commons blogs with date 16th for December 2016 which is written by Dr Emma Shtivelman. This text is describing without “saying” the analysis that is offered to cancer patients from RGCC SA.
Well, after reading very carefully her opinions in this text I found many (if not all) points of her claims that are completely unrealistic and lack of scientific background (despite the title that the author has obtain).

In Europe, we cannot make these types of claims without scientific evidence. I will list the main points that Dr Shtivelman raised to support her claims, under scientific evidence and evaluation.

  1. Dr Shtivelman wrote “This cautionary tale begins with one of the company’s advertised tests, which promises to provide results on CTCs for any type of cancer.”- This is not true since the analytical method that is used for the first step of analysis is the isolation of CTC and there are types of malignancies (like primary brain tumors etc.) that cannot be detected and isolated. So in this case we can conclude that Dr Shtivelman made a clear assumption without any scientific search about the analysis itself.
  2. Dr Shtivelman wrote also: “Testing for BCR-ABL is DNA-based (proteins are not generally used), and the reasons for doing it for solid tumors, such as lung or brain tumors, are not clear.”- Again, we must say that this is also an assumption. First, the analysis of BCR-Abl is a test in mRNA level (cDNA converter from mRNA), and second the analysis is the third step after isolation and cell expansion which uses all genome gene expression hyper-dense micro-array. This means that the analysis is performed on the mRNA from all genome that is expressed on the cancer cells. We never claim that the analysis is based on proteins. And again, Dr Shtivelman wrote “The important part of this test, ostensibly, is the analysis of several proteins known to be present in some selected cancers. I should say, “randomly selected proteins,” but more on that below.” So, again it becomes obvious that Dr Shtivelman made a very serious incorrect assumption about the analytical method that is used by RGCC.
  3. Dr Shtivelman also wrote: “Here is the first major problem: CTCs are found in the blood of most patients, especially those with metastatic cancers, but they amount to no more than 1 to 10 cells per milliliter. With extreme precautions, CTCs can be isolated from a fresh blood sample if they have the protein EpCAM, which serves as a “tag” that allows them to be captured and pulled out of the sample. However, far from all cancers express EpCAM.” To this point, it is obvious that Dr Shtivelman ignores all the methods that exist for the isolation of CTC (like the size based method using gradient density material or membranes etc). She also ignores that our methodology is a negative based method that is performed in a flow cytometer and sorter (accredited and certified method based on ISO 17025) this subtracts the blood origin cells and leaves the rest of the cells without stain and immobilization (hence still viable). I strongly suggest Dr Shtivelman and the rest to read the book “Minimal Residual Disease and Circulating Tumor Cells in Breast Cancer” Ignatiades et al. You will find all these techniques described there and not the narrow and limited methods that Dr Shtivelman describes. In addition, Dr Shtivelman ignores the process of EMT, which the epithelial origin cancer cells transit their phenotype to a mesenchymal one which downregulates all epithelial antigen including EpCam.
  4. Dr Shtivelman also wrote: “Because it is not possible to analyze them in the first place, the bizarre selection of proteins that this company has chosen to analyze is definitely a secondary issue.”. Again, Dr Shtivelman makes a false assumption that we select the proteins as it is written above, the analysis that is performed is on all genome expression micro-array and not protein analysis.

Dr Shtivelman also wrote: “The fact is, CTCs are notoriously unstable and cannot be cultured, with very rare exceptions.” Again, I address you and Dr Shtivelman to the literature like Yamamoto et al “Ex vivo culture of circulating tumor cells using magnetic force-based coculture on a fibroblast feeder layer”. For any expert, it is obvious that the CTCs need to be treated like stem cells and not as a typical cell line, which is the major method most labs use. In an extensive search, there are plenty articles and books describing how CTCs can be cultivated and their limitations. Further reading of Kulasinghe et al in “Short term ex-vivo expansion of circulating head and neck tumour cells.“

Dr Shtivelman also wrote: “Let’s do some simple math: well-established cell lines (immortal cultures of cancer cells grown in labs all over the world) double in number in one day or so.”. Again, Dr Shtivelman confuses cell lines with primary cell cultures and stem cell culturing kinetics, which there is no relation between them. It is a clear assumption that the doubling time is 24 hours since the growing rate of CSCs can be up to many thousand times faster than a typical cancer cell line. See relevant article Toloudi et al 2014 “Comparison of growth curves of cancer cell lines and cancer stem cells”.

Dr Shtivelman also wrote: “This company fakes all the results in their reports and shamelessly charges patients a handsome amount (over $3000) for tests that are never conducted.”. This is the point where Dr Shtivelman not only made a false assumption, but also made false accusations for pricing. Pricing is transparent for all areas served by RGCC and the price is in Euro not in dollars. and it is not to that level. Also, I need to familiarize and remind you that RGCC lab facilities is supervised by ESYD which is the state representative of IFA (International Forum of Accreditation) and all our protocols are accredited by ISO 17025. Also, RGCC is under the assessment for the same assays of EMQN (European Quality management network) which sends us blind samples and we must send the analysis back to them and be on standard z score of accuracy. Finally, since we preserve all our data, the isolated cells and cDNA we can send the material to any other third lab to confirm the data. So I really wonder under which criteria and evidence Dr Shtivelman made all these false accusations without a visit to our facility or even took the time or courtesy of simply contacting us?

Dr Shtivelman also wrote: “Safe to assume that, based on test results, the poor patients will receive the highly popular and expensive intravenous injections of vitamin C, or will be rushing to purchase one—or, likely, more than one—of the “natural substances” with exotic names like “Ukrain” or “Okinawa extract,” etc.”. Once again, Dr Shtivelman made another false statement without any evidence, since the test result does not show which vitamin C had to be obtained. The cells are assessed in micro- culture of 12,000 cells/well with ascorbic acid that is obtained as a chemical substance from chemical supplier. Also, natural extracts “with exotic names” like Ukrain or Okinawa extracts are commercial names of substances with biological effect. To be precise, Ukrain stands for the chemical chelidonine and Okinawa extracts is an extract of Fucoidan with a very long literature history of anticancer activity. Also, we must not forget that many natural agents have been used in drug development on a ligand based or structure based drug design including several chemotherapeutics.

We conclude on the below points the obvious false assumptions and inflammatory remarks made by Dr Shtivelman:

  • She made many wrong and false assumptions (we do not know if these were intentionally or unintentionally)
  • She never read our web site text , disclaimers, data etc
  • She never contacted us for information about the analytical methods that we use.
  • She didn’t use any evidence based science with her remarks
  • She raised doubts and made accusations without any evidence of proof and without any scientificreference that would accurately reflect our methodologies.
  • She spread very negative rumors which lack no evidence and they are based only on her personal

opinions.

• She used unscientific assumptions that mislead readers to false and possible harmful outcomes.

As you must realize, we cannot let these unresponsibly and likely harmful personal assumptions go without responding. Since your network tries to help patients based of expert opinions, this is not the responsible way to help your readers. We are asking from you to reestablish our damaged image and reputation that Dr Shtivelman has caused. We also ask directly from Dr Shtivelman to withdraw her comments and put a public apology about her false and unscientific accusation about RGCC SA. Otherwise, we will be forced to proceed toward legal actions for all the above false and misleading statements (which are reaching the low level of gossip).

Hopefully this blog will regain a creditable reputation by honoring the truth that I have now made you aware of so legal action will not be necessary against the blog owner, personnel and/or Dr Shtivelman herself.
I look forward to your reply as soon as possible on this last issue.

Sincerely,

REPLY

Sent from my BlackBerry 10 smartphone.

From: Emma Shtivelman
Sent: Παρασκευή, 27 Ιανουαρίου 2017 – 19:20 To: Dr. Ioannis Papasotiriou
Subject: RGCC tests

Dr. Papasotiriou,
I have drafted a very detailed response to your email, and it is now in hands of our legal council.

I would like, however, before the matter is transferred into the legal sphere, to ask a few questions and clarify some important points.

To start, you are well aware that the blog post on Cancer Commons website does not mention your company by name, so the legal value of your intent to take legal action is extremely dubious. Moreover, there are sources on the web that are extremely negative about the RGCC services, and your company is certainly named there. I am not sure if you are aware of that.

My goal in writing this post was not to cause offence (this is why RGCC is not mentioned by name), but to protect cancer patients from spending money on tests that are not clinically relevant and are not at all validated for clinical use.

I would like to ask you therefore what is the scientific basis of your claims. The most important questions are: first, what is the basis of your claims that you can isolate live cells from blood collected days earlier? In all the many publications about CTC isolation for culture blood sample are processed within HOURS of venipuncture, with a good reason (gragility of CTC). What is your rate of successful CTC isolation?

Second, RGCC claims that it is able to propagate hundreds of millions of cells within days from the very few that it isolated from 20 ml of blood. I have dutifully collected numerous reports where culture of CTC was documented, and none of them claim to be able to culture cells from all samples collected (success rate varies

from 3 to 20%). See also https://www.ncbi.nlm.nih.gov/pubmed/25998619 for references to the propagation of CTC ex vivo. In particular, here is the conclusion of this review written by the leaders in the field:
“While CTCs presumably include metastatic precursors capable of initiating distant lesions, most of the tumor cells isolated from the bloodstream even using gentle microfluidic conditions are not viable”

More importantly, even if successful, propagation ex vivo produces only a small number of cells even after 2 weeks of culture. In particular, Kulasinghe et al (https://www.ncbi.nlm.nih.gov/pubmed/27517751), the paper you have cited in your email, describes that CTCs were isolated from 56% percent of HNSCC patients (a little more than half), and of these that were successfully isolated, only 28% produced cultures. This amounts to a success rate of ex vivo propagation of 16%. Moreover, it took at least 2 weeks (and often more) for cultures to reach semi-confluence in 96 well plates, which means that after 2 weeks investigators were able to propagate perhaps a few hundreds cells.

How would you compare these published data (cited by you) to the success rate seen in your laboratory, with millions of CTC obtained in a few days? This, by the way, means a doubling time of anywhere between 3 to 7 hours, an unheard rate for adult cells, stem or not.

I would appreciate if you will address these very serious questions about the RGCC claims. I am ready to engage in a discussion, including a public one, regarding the validity and clinical usefulness of RGCC tests. You may have an opportunity to have your respons posted on the Cancer Commons website, for example.

Looking forward to hearing from you.

Sincerely,
Emma Shtivelman CancerCommons.org

The information in this email has not been provided by a physician. It is intended for educational purposes only and is not a substitute for treatment or advice from a physician or healthcare provider.

1.27.17 response

Dr Shtivelman,

I just receive and read your reply. As it seems from a brief read several other publications are ignored that they manage to isolated and expand CTCS (from lung carcinoma cases) in the percentage of 84% (Ann Arbor). These are similar rates of what we achieve on our lab. Also, you ignored that the isolation method that we use is negative selection and you introduce methods of positive selection as a comparative technique and finally you ask about the expansion methods to justify why others cannot grow at all or very poorly the CTCS when already I’ve mentioned to you that CTCS should be treated as tumor initiating cells and not as cancer cells lines.

So, after my return from my trip back to my home (approximately in the middle of next week) I will reply back with peer review and broad spectrum peer review literature.

To that point, I have mention that by introducing selective articles and publications and ignoring serious points and evidence defeats the goal of helping patients. On the contrary such comments and claims are misleading patients and you may cause more confusion instead of clarifying those grey areas on cancer treatments. Hence, I must insist that my original conclusions and claims that such kind of misleading comments are subject to legal actions in Europe because our regulations are much stricter than on the other side of the Atlantic. Finally, keep in mind that starting in 2017 we have decided as a company to take a more offensive stance against those who are making similar claims against our company. Therefore, the excuse you made that others make worse comments than yours will not stop our new strategy in the future.

I will reply when I am back home.

Sincerely,

Dr Ioannis Papasotiriou MD PhD, C.Cy, QP Director of RGCC SA

Qualified person (QP) is a technical term used in European Union pharmaceutical regulation (Directive 2001/83/EC for Medicinal products for human use). The regulations specify that no batch of medicinal product can be released for sale or supply prior to certification by a QP that the batch is in accordance with the relevant requirements.(EudraLex, Volume 4, Chapter 1) The QP is typically a licensed pharmacist, biologist or chemist (or a person with another permitted academic qualification) who has several years’ experience working in pharmaceutical manufacturing operations, and has passed examinations attesting to his or her knowledge. The requirement for QP oversight has been extended to material for use in clinical trials since the introduction of EU Directive 2001/20/EC.

1.31.17

Followup response from above.

Dear Dr Emma Shtivelman,

As I promise in my last email I would reply in detailed to your last email. Let’s start with the scientific-medical issues.

From the total 8 points that you raised in your original article on December 2016 you narrowed those down in your last email to the below major points:

  1. How it is possible to isolate the CTCs and keep them viable.
  2. How it is possible to cultivate them and generate sufficient number of cells for further analytics.

The above points have been supported from articles that you have sent to me by a link from Pubmed. To the first point I read the reference article and we have to mention the following points:

  • It is a literature review article and not a scientific or medical one (it assesses methods without actually put them on a lab bench test).
  • It has narrowed down the perspective only to methodologies of positive selection based either on CellSearch technologies (a very well-known method with very limited sensitivity) of a chip technology with passage from a micro-capillary with antibodies for cells selection.
  • Also, in the description of the techniques all the methods described by FACS described the depletion of RBC and WBC. But, what about the endothelial cells and pericytes that also exist in a regular blood sample even if you discarded the first 5 to 10ccs? So, I must assume there were also epithelial origin cells there as well.
  • None of the positive described methods that the appointed article described took into consideration the cells that are in mesenchymal phase (those are lacking the epithelial antigen including EpCam).
  • Also, all these techniques have no reference about the portion of CTCs which are fused with monocytes and by the described method the fused CTCs are already depleted.So, already the review article has many and very large gaps on the actual description of methodologies about isolation of CTCs.

    The methodology that we use after assessing many assays for isolation of cells as rare events, is a flow cytometry based on negative selection. Since the original blood sample has been treated we deplete the RBC and platelets and then the cells are interrogated by depleting WBC and endothelial cells (using CD31 or PeCam) the droplets are adjusted in one event per one droplet. The inner chamber is regulated so that the core pressure is low so that cells will be viable and in a single cell suspension. Then the isolated cells stay viable. Since the method has been assessed for LoD and LoQ the values are 1.4 cells/10^7 events (WBCs) and the LoQ is 4.5cells/10^7 events (WBC). So, this is an actual rare event method since rare events are defined by the ratio of 1:10,000 . So far most of the described methods in the review article do not mention LoD and LoQ and even those that they do have are at in much higher levels.

    Now, let’s look at the second point that you have raised about the possibility of cultivating the CTCs. The controversy here is that the article you are referring to mentions a rate of 56% success in cultivating, the reference 30 of the first article that you printed for me is describing expansion of CTCs in 16 cases of Lung cancer of the 19 (rate approximately 84.2%). So, we already start with a controversy of what you stated based only on your references. Also, the attempt of Dr Zhang, in the reference is on track of expanding the cells treated as Cancer stem cell like and this is what makes the difference. Also, in your text in the web site you mention that the doubling time of CTCs is on average 24 hours and on your second text the doubling time was defined in the level of 3 hours. Well, I mention an article of Toloudi et al of 2014 which the Cancer stem cells are grown and compared with cancer cells line and it is show the following features:

    • There is no or very short lag phase (adaptation phase)
    • The log phase of the cultures between CSCs and Cancer cell lines may have a difference of growingfrom 100 to 10000 times. Hence, CSCs have variable growth rates than the typical clonal cancer cell line or immortalized cell line.

      So, the doubling time is much shorter that the limit of 3 hours. So following your example if at the end we have 25ml of whole blood with 4cell/ml then in total we have 100 cells with doubling time of 1 hour we conclude with a number of 1.6 billion cells in the first 24 hours.

So, the following becomes obvious:

The references that you have used are controversial with your statements and finally I suggest to read more carefully your reference completely and even better, to test them on a lab bench before you take them for granted.

Now, let’s go back and review the points that you raised in your primary statement:

1. You stated that out test is for all kinds of tumor- THAT WAS A CLEAR FALSE ASSUMPTION SINCE THERE ARE LIMIATIONS AND BARRIERS

2. You stated that we analyze the protein profile of the cells- THAT WAS AN INNACCURATE ASSUMPTION SINCE THE ANALYSIS IS BASED ON THE GENE EXPRESSION PROFILE WHICH IS AN OUTCOME OF MICROARRAY

3. You stated that we use positive selection method based of EpCam antigen- ALSO A FALSE ASSUMPTION AND INNACCUARE STATEMENT SINCE OUR METHOD OF DETECTION AND ISOLATION IS BASED ON NEGATIVE SELECTION

4. You stated that we put fees of 3000 USD- THAT IS A FALSE SPECULATION SINCE WE HAVE A TRANSPARENT PRICE POLICY AND THE PRICES ARE IN EUROS AND NOT TO THAT LEVEL ON ANY OF OUR TESTS

5. You stated that we test exotic products- THAT WAS ALSO FALSE SPECULATION AND ACCUSSATION SINCE WE TEST ONLY PRODUCTS THAT WE CAN REVIEW AND OBTAIN AS PURE AGENTS AND MANY OF THOSE HAVE BEEN ASSESSED AND EITHER DID OR DID NOT PROVE THEIR EFICACY.

From all the above it becomes more than obvious that:

You use reference literature and by manipulating their outcome you cause a misleading outcome to the readers of your articles. Also, it would be a very good challenge if you come and visit our facilities and see all the above in the lab in real action and not by reviewing a review selectively. Then it would be interesting to hear your comments after that.

So, I have to have to maintain the same position stated on my first email which was:

· You made many wrong and false assumptions (we do not know if these were intentionally or unintentionally).

  • ·  You never read our web site text, disclaimers, data etc.
  • ·  You never contacted us for information about the analytical methods that we use.
  • ·  You didn’t use any evidence based on science.
  • ·  You raised doubts and made accusation without any proof or evidence and without any scientific reference that reflects our methodologies.

· You have spread very negative rumors which lack any evidence and they are based only on your opinion only.

· You use unscientific assumptions that mislead your readers to a false outcome.

So, if this is not bias then what is? I must also point out that if your intentions are to help patients, you do not help them by falsifying reports, articles and making false assumptions which are presented as objective findings.

Such behavior is far from a scientific one and even more, not even medical (but from that you are excused since you are not a medical physician).

Going now to the legal issues. Bear in mind that since your comments are publically reported through internet this provides us with the privilege to raise a legal issue in the regulated environment of Europe. Therefore, the lack of mentioning our company name is not sufficient to avoid such an incident here in Europe.

So, unless I receive a public apology for the false accusations and wrong assumptions, I will have no other choice but to proceed immediately to the next step of legal actions with no further notice.

I will wait for your reply until the end of this week. On behalf of RGCC SA

February 10, 2017
Dr. Ioannis Papasotiriou R.G.C.C. S.A.
Industrial Area of Florina, Plot 4 GR 53100 Florina, Greece +30-23850-41960

Dear Dr. Papasotiriou:

My name is Marty Tenenbaum and I am the founder of and Chairman of the Board of Directors at Cancer Commons. I have been apprised of the circumstances and ongoing correspondence between yourself and Dr. Emma Shtivelman regarding her December 16, 2016 blog post, titled “Beware of Predatory Cancer Testing Companies” (https://www.cancercommons.org/category/knowledge-blog/lung-cancer/).

Second, I am more than happy to engage with you in further discussions regarding your lab, your research and your work. I believe it would be very beneficial for me to better understand your lab’s processes, methodologies, etc. We all have the same goal of assisting cancer patients and their families, and I believe that continuing open dialogue amongst members of the global scientific community is the best way to accomplish this.

While I appreciate your offer to visit your lab, unfortunately, right now is not convenient for myself or my staff to make the trip to Greece. But I would like to extend an open invitation for you to come and visit us here at

First, I wanted to personally reach out to you and let you know that Cancer Commons has taken down Dr.

Shvitelman’s blog post.

Cancer Commons the next time you would be traveling to the United States. Likewise, I would like to come and visit you and your lab when my schedule permits.

Please feel free to contact me at your earliest convenience to continue this conversation. My phone number is (650) 799-1767, and my Skype handle is marty_tenenbaum. My assistant Masako Yokota (cc’d above) can assist with scheduling. I look forward to hearing from you.

Very Truly Yours,

Marty Tenenbaum, PhD
Founder and Chairman, Cancer Commons

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