22 replies to this topic

[valiantchong]

Question for the board:

1) Does RT-PCR able to detect translocation of BCR-ABL chromosome in quiescent Leukemic cells or stem cells

2) If RT-PCR method could be use to detect translocation chromosome in quiescent leukemic cells, does that means if someone achieve PCRU there will be no more LSC or the RT-PCR is not sensitive enough to detect them ?

3) Since every cells have chromosome (even in stem cells) does that mean quiescent leukemic cells or stem cells chromosome translocate only of after reactivate ? Any other diferent in chromosome between LSC or normal stem cells ?

I have been wondering why someone in PCRU who relapse is due to quiescent leukemic cells reactivate or due to new cells translocation ? If it is due to old stem cells reactivate, then why RT-PCR fail to detect these quiescent stem cells. ? Is it because it is too small in quantity or not sensitive enough for RT-PCR to detect the quiescent stem cells ?

Anyone or Trey could enlighten me on this, I am deeply appreciate... thank you.

[PhilB]

The only way you'd detect a higher level quiescent stem cell in a PCR would be if you actually happened, by some million-to-one chance, to hoover it up the needle in a BMB.  If the stem cell is quiescent ie not reporducing then you have to find that exact cell to pick it up on a test.  If it isn't quiescent then you can detect any of its millions of downstream descendants.

As to whether measurable PCR following PCRU is a new translocation or a waking up, that depends on what model of CML you believe in.  Some people argue that the translocation event (in a high enough level stem cell) is rare and therefore the odds of getting another one would be very low.  Others argue that the the translocation is common and it's our bodies' inability to recognise and destroy it that is rare.  In reality though, in most cases of positives following PCRU the real disease level was probably never zero, it was just below the level of sensitivity of the test.

[Trey]

1) Quiescent, no.  Non-quiescent stem cells, yes.

2) At initial PCRU ("barely PCRU") there are still about 1 million leukemic cells in the body.  Deeper PCRU levels (below sensitivity of PCR) can be as low as 1 or none (none = cured).

3) Leukemic cells come from other leukemic ells, except for the first translocation.  So they all have the Ph+ Chromosome as they sit in the quiescent state.  When they divide the new cells are all leukemic.  A leukemic cell cannot make a non-leukemic cell.

4) Relapse is due to low levels of undetectable leukemic stem cells.  Even a transplant can relapse when the quiescent cells are not killed off, and GVHD also fails to destroy them.

[valiantchong]

Hi Trey,

Thanks for your reply, however you answer on 3) they all have the Ph+ Chromosome as they sit in the quiescent state. ? I have another question, if all cells have Ph+ chromosome as they sit in quiescent state, why RT-PCR could not pick them up ??

If the quiescent cells' chromosome could detected by RT-PCR then how could we know what is the quiescent % vs non quiescent % ? Any test able to differentiate that :?

appreciate you answer.

Thank you.

[Trey]

In the quiescent state they hide deep in the bone marrow stromal layer where they do not circulate in either the blood or marrow fluid.

[valiantchong]

Hi Trey,

My next question is how could a quiescent state cells hide and move towards the bone marrow stromal layer, since LSC queiscent cell and normal cells do not have special mobility inside the blood plasma. How could these cells move towards a certain known direction and stays there compare to other non-quiescent cell ?

[Trey]

Stem cells have special homing instincts.  It is a survival mechanism.  They are very good at surviving.  Most of the time that is a good thing for the organism.  In this case, it is not a good thing.

[valiantchong]

Hi Trey,

Thank for the reply, I tried to find your email address but not listed in your profile. Hence I would ask in this board, wondering do have any write up article on the LSC homing ability to move to the bone marrow storma niche.

As I know there are article saying the quiescent LSC are most probably CD34 + and as time goes on these CD34+ will divide, and eventually these will hit by TKIs. There are an article stating with GF these CD34+ cells divide even faster and hence TKIs will kill them too.

I believe after sometime these pH+ CD34+ cells will die off until RT-PCR will not be able to detect them, that is why most of the PCRU patient after years about 40% could stop TKIs without any relapse. Time will tell if this is true or may be the T cells kick off and able to control these MRD ph+ cells in equilibrium.

[Trey]

It is not quite so easy.  The highest level leukemic stem cells are higher than CD34+, which are actually just progenitor level cells.  No one knows what the very top of the leukemic cell chain is for certain.  The stats you cite do not account for the increasing difficulty of killing the leukemic cells above CD34+.

Here is a good article about leukemic cells homing to the marrow niche (although focused on AML):

http://bloodjournal....114/6/1150.full

[valiantchong]

Thanks for the diagram of LSC information below, the LSC may differentiate with CD34+/CD38- murlin lin- cKit+ and Sca-1, base from the articles I understand they acquired adhesion molecule as VLA-4 which make them adhere to the bone marrow. All these LSC are Ph+ cells, will eventually divide, when this happen TKIs will be ready to hit them unless they aquired immunity or resistance against TKIs drug. I believe from statistic point of view of survival of the fittest, will push out all these cells after a few years, from mathematical model, it may be 2-5 years time, if one is lucky and hence be 'cured'. This is an  open discussion.

Since CML only affect myeloid cells and Not Lymphoid cells, I believe CML Stem cells could not be so high up as to effect the highest HSC which will differentiate to Myeloid Stem cells and Lymphoid Stem cells. Hence CML stem cells will only be concentrate on myeloid stem cells after HSC differentiate. These LSC could be identified and characterized by the leukemic cell's surface of CD34+/CD38- murlin lin- cKit+ and Sca-1. There are article stating all SCs inculding LSC will divide in after 5 to 6 cells cycle. Calculating our blood cells divide daily, the population of LSC will eventually push out. On the positive side, our T-cells will also help kills these cells after TKIs improve our immune system.

[Trey]

VC,

Your statement: "CML only affects myeloid cells and Not Lymphoid cells" is not accurate.  CML may affect the myeloid line to a larger degree, but the lymphoid line also has the Ph+ chromosome.  So your theory about the level of the CML leukemic stem cell being lower in the cell line is inaccurate.  The article you cited is about AML, not CML -- I suggested you read that article because it had the best explanation of leukemic stem cell homing that I could find, even though it was about AML.  The AML originating leukemic cell may be lower in the cell line than the CML one.

It is also not known that when leukemic stem cells come out to divide that TKI drugs will affect them.  In vitro the TKI drugs do not affect the highest level leukemic stem cells at all.

[scuba]

Trey - take a look at these two papers in sequence (they are fairly recent):

1:  http://www.scienceda...01213121704.htm

2:  http://www.prohealth...cfm?libid=15529

Curtailing the beta-catenin pathway affects leukemic stems cells and as reported in the first article causes the cells to revert to a pre-leukemic stage.  I do not know what they mean by pre-leukemic?  Does the PH+ chromosome repair itself?  or is it just inactivated.

It is believed that the Curcumin I am taking (dose dependent) is doing this (Dr. Aggarwal) and causing the LSC's to not replicate and replenish the population.  My dramatic PCR level drop as confirmed by M.D. Anderson has now caught the attention of the research team.  Dr. Cortes has contacted me and asked that I submit to a workup involving documenting the Curcumin I have been taking.  They will do this in early November.  If my PCR shows a  3-4 log reduction or even PCRu by that time - he may want to do a study with Sprycel + Curcumin for CML.  I don't know what the protocol is for FDA considerations because Curcumin is a naturally occurring compound and there is no restriction.  Could he ask patients to add Curcumin (8 grams by the way!) to their daily routine on his own? - and then just track results on his low dose patients?  Recall - he uses low dose to manage myelosuppression.

None of this is confirmed until my PCR in November.  That will inform him - but I do have his curiosity.  No one has had the drop in PCR I have experienced on 20mg Dasatinib.

It's been one month post my PCR results/test with another two months to go before I go in to M.D. Anderson for the all day affair including bone marrow.  I have had no CBC done for weeks now (no need to according to the docs) but I do feel my blood levels are improving simply by how I feel (no more dry mouth - no sores whatsoever - no petechiae - energy back up (no anemia?) and no more fatigue).  If it wasn't for the fact that I "see" the Sprycel bottle on my night stand and take it every night - I would not know I had CML.  The Sprycel pill is so small I often don't feel I have swallowed it.  There are no side affects. (Oh - no correlation between drug amount and cost.  20mg. is only a bit cheaper than the 100mg version).

What I believe you would be most interested in is that Dr. Cortes is very interested in TKI dose studies.  Like you - having to take max dose may not be necessary to achieve excellent results.  He is looking for a way to increase effectiveness of treatment while lessening side affects (either with new compounds or with added compounds like Curcumin).  I am already part of a phone-in study once per week on side affects with his lab (they call me and I answer automated questions on how I feel - this has been going on for about 10 months).  His prescribing my current dose went against the recommendation of my primary Oncologist who wanted me at 100mg Sprycel and then take stim shots.

If a TKI dose can be lowered in conjuction with some other non-toxic compound - this may be a path for improved quality of life for many people.

If it turns out that my PCR is not changed or otherwise results are not signfiicant - then Curcumin is just a good anti-oxidant for your arteries as is what most people take it for.  We'll see.

[valiantchong]

Hi Trey,

I have not come across any article stating CML in CP will affect Lymphoid cells unless it progress or mutate to ALL. If you have any article on this, pls share as my knowledge in this area is still green.

Thank you, 

[valiantchong]

Hi Micheal,

I read your forwarded article on DNA repair, below was another example. However I do not know why there is no indept study on this, I undertand vitamin B complex, if I am correct B-17 has ability to help in DNA repair... any other article could show this is another pathway to cure ?

Cell lines provide insight into DNA repair in leukemia

Horizon Discovery, a provider of research tools to support the development of personalised medicines, has announced that it has secured a worldwide exclusive license for 50 new X-MANTM (gene X- Mutant And Normal) cell lines from Public University Corporation Yokohama City University (YCU).

The panel of cell lines, which includes many important double knockout cell lines, is focused on DNA repair and is in the NALM-6 cell line, a B-cell derived line. Therefore, the agreement extends Horizons panel of X-MAN cell lines to include B-cells.

NALM-6 is a human B cell precursor leukemia cell type, established from the peripheral blood of an acute lymphoblastic leukemia. These new X-MAN cell lines will allow researchers an insight into the role of DNA repair in leukemia, as well as providing a model for other diseases. The new panel also complements Horizons existing X-MAN DNA repair panel in the HCT-116 colon cell line, providing extensive reagents for the analysis of DNA repair pathways.

Under the terms of the license agreement, Horizon will pay YCU up-front and on-going royalty payments, in return for world-wide exclusive rights to distribute these lines.

The agreement forms part of Horizons strategy to generate at least 2500 new X-MAN models of cancer, neurodegenerative, and cardiovascular disease. These models will support drug discovery researchers in their effort to understand how complex genetic diseases manifest themselves in real patients and help rationalize many aspects of drug development, reducing the cost of bringing to market new personalized therapies.

Dr Rob Howes, Principal Scientist at Horizon, said: We are pleased to license these cell lines from YCU as this extensive panel of single and double mutants in genes involved in DNA repair will allow researchers to understand the mechanisms of DNA repair and how they are involved in the development of leukemia.

For more information, visit www.horizondiscovery.com

[Trey]

VC,

I think you answered your own question.  CML could not progress to ALL in blast phase unless the Ph+ was already in the lymphoid cell line.

[Trey]

Michael,

Since neither of the articles you asked me to look at are about CML, it is hard to draw any conclusions from them about CML.

[scuba]

Trey,

I was extrapolating subjectively.  Nevertheless, here you go:

http://www.ncbi.nlm....pubmed/18068630

[Trey]

You should not extrapolate subjectively in public.  Just sayin'

Regarding the most recent link, it suggests that altering beta catenin pathways (by curcumin, I assume you are saying) might possibly impact both leukemic and normal cell production, suggesting it would also make your anemia worse.  If your anemia is not getting worse, then maybe the curcumin doesn't affect either normal or leukemic cells.

[scuba]

Trey,

It's a 'down regulation' process.  I suspect "subjectively" that both cells are affected as you point out - but Leukemic cells exhibit accelerated growth.  Down regulating the beta-catenin pathway most likely affects them more than normal cells.  It is a population dynamic problem.  For example - TKI's affect normal ABL also - hence our side affects.  But it is a question of what is affected 'more' - and balancing both.

And it's not just beta-catenin that is down regulated.  NF-kappaB is down regulated as well and is known to be important in Leukemic stem cell replication and is not required in normal hematopoietic stem cell function.

from: "http://www.ncbi.nlm....les/PMC1988840/"  The Leukemic stem cell:

"Normal hematopoietic stem cells do not show activation of NF-?B. We believe that this is a leukemia-specific phenomenon." 

Shut down the NF-kB pathway and Leukemic Stem cells get unhappy.  Curcumin downregulates that pathway.

I experienced a long period of slow recovery following TKI cessation while taking Curcumin.  So I suspect you are correct "subjectively" that Curcumin slows things down.  For Leukemia - this slow down is detrimental to the process and is dose dependent.  And as long as the Sprycel can get the upper hand - I am pleased.

In reality, Trey - we know Curcumin is a benefitical 'herb' at normal diet doseages.  A long history of use highlights its beneficial affects (nutritional).  What I am doing is upping the doseage to a higher level based on the dose dependent evidence that Curcumin intereferes with programmed cell death (restores) and Cancer (B. Aggarwal, M.D. Anderson) and "testing" whether I receive a beneficial response.  Not a cure - it just seems to help drugs do their work (check out Curcumin effects on Myeloma).

Subjectively speaking - I extrapolated from the Colon cancer studies to CML.  I look forward to the November tests and the liklihood that M.D. Anderson wants to take a closer look ...

"objectively".

[valiantchong]

Hi Trey,

Which means CML in CP there will be no Lymphoid affected unless it mutate or progresses.

Hence the theory of CML boundary exist only in Myeloid cell is correct. If Ph+ in CML affect Lymphoid  then all CML will progresses to ALL, but there is only a hand full of people that progress to ALL, which means CML in CP will only affect Myeloid cells..