Multiple Myeloma - Science topic

The exact ng will depend on the expression of your genes in your exact samples. You can check publications with similar experiments to get an idea of the range. But the only way to know for sure is to try your samples. The kit has suggestions for how much to start with.

You'll have to know something about the lower detection limit from your standard curve. You can increase the amount of starting ng for your genes-of-interest and account for the change in starting materials for your calculations.

Good luck!

Dear all

This is the latest info I have - from a review article in Seminars on Oncology (ahead of print):

”Whole-body low-dose Computed Tomography is now recommended over the conventional skeletal survey, and more sophisticated functional imaging methods, such as 18F-Fluorodeoxyglucose Positron Emission Tomography , and diffusion-weighted Magnetic Resonance Imaging are proving effective in the assessment and monitoring of MM disease.”

Dnase I from Qiagen.

Anger, aggressive behaviour can often lead to headache,poor digestion leading to abdominal pain,insomnia,increased anxiety or depression.Raised blood pressure,skin problems, such as eczema and heart attack.

Evidence show that suppressed anger can be a precursor to cancer development and also a factor in its progression after diagnosis.

Stress also linked to cancer progression and hard to treat.

However,Long term studies are needed.

Too early and naive for definite answer.

The survAUC R package provides a number of ways to compare models link: https://stats.stackexchange.com/questions/181634/how-to-compare-predictive-power-of-survival-models

Hi Bikash

The plaques could be amyloid (secondary to myeloma) deposits. You may want to biopsy them. If the suspicion is confirmed, ideally the patient should have a SAP scan. As to treatment, the choice would be a velcade based combination. (eg VCD).

Best wishes

Siva

First - do you want to overexpress some gene/protein or go for gene silencing?

For the latter one, especially when using siRNAs I never faced any problems while using the electroporation method in any Multiple Myeloma cell line.

But if you want to insert huge plasmids, the situation is very different...

Good question. We have to wait the results of the research work in the future.

It will depend on the future study that you have to conduct with your cells.

Hello!

I find some publications about knock down CD38 in multiple myeloma

Maybe this articles will help for you?

Thanks Lael Werner and Laurence Stuart Dawkins-Hall very much for your helps.

Please keep one thing in mind when you work with primary MM cells. Plasma cells are terminally differentiated, so the basic thing they will do is to die, but they are not supposed to really grow any more.

Giving them a good environment, especially seeding them on stroma cells slows down the apoptosis speed, adding IL-6 usually also helps, but to a worse extend than stroma cells. We seed the primary cells in RPMI1640 medium enriched with 10% FBS, 2 mM L-Glu, Pen/Strep 1%, 1mM Na-Pyru.

This is one of the oldest papers from the group where I am working, around 20 years of experience with primary cells, still we use the protocol today.

In brief,

1. Use the genomic sequence to find out potential transcription factor binding site(s) using the TRANSFAC program (or any other free program);

2. Generate a reporter construct with the promoter sequence to be tested;

3. Use potential sequence(s) for a candidate TF to perform binding assays using the labelled cis-sequence;

4. Mutate the potential TF binding site(s) in the reporter to demonstrate the specificity of binding;

5. Perform ChIP assays to demonstrate binding of a TF to cis-sequence in vivo (in cells).

Frank Arguello Thank you so much

Thanks very much Robert. I really appreciate it. I am also wondering if have ever observed the death of a large population of RPMI 8226 cells if you seed them in low concentration.

Thanks.

Dear Monika,

The exact diagnosis of your father is probably smoldering multiple myeloma (SMM), according to the information you gave about his medical data. SMM is defined as following criteria:

Serum monoclonal protein: IgG or IgA ≥3 g/dL, or

Bence Jones protein ≥500 mg/24 h  and/or

Clonal bone marrow plasma cells between 10% and 60% and

Absence of myeloma-defining events or amyloidosis.

The risk of progression to symptomatic multiple myeloma of SMM is about 10% annually.

Experts in hematology consider SMM as an excellent setting to test the efficacy of novel theurepatic drugs such as ixazomib, pomalidomide, elotuzumab, and daratumumab in myeloma treatment.

Despite standard care of SMM is close clinical observation until development of symptoms, International Myeloma Working Group recommends therapy for SMM cases at high risk (about 50% risk for progression to symptomatic multiple myeloma).

For more information please visit:

Yes..

Yes, you should evaporate ethanol completely. Any trace amount of ethanol could affect the quality of RNA.

Good luck!

Will try DMSO, thank you Saganuwan Alhaji Saganuwan Samir G Pandya

In normal person detection of plasma cell as an reactive cell especially in infective or inflammatory process is possible

Daratumumab plus pomalidomide and dexamethasone in relapsed and/or refractory multiple myeloma

Ajai Chari, et al. Blood 2017 :blood-2017-05-785246; doi: https://doi.org/10.1182/blood-2017-05-785246

Clinical Efficacy of Daratumumab, Pomalidomide and Dexamethasone in Relapsed, Refractory Myeloma Patients: Utility of Retreatment with Daratumumab Among Refractory Patients

Ajay K Nooka, Nisha Joseph, Larry H Boise, Charise Gleason, Jonathan L. Kaufman and Sagar Lonial Blood 2016 128:492;

Hi Zoabi, I am wondering about how to extract myeloma-associated macrophages directly from the bone marrow, and I saw that you had posted this similar question a while ago. Did you find an answer to this? Best, Kristian

Hi Santa,

I would go with Staining Index or Separation Index (thats takes into account Positive and negative population values of Fluoresence), I join you this very helpful mini-review-blog that links to the fundamental articles: https://expertcytometry.com/stain-sensitivity-index/

After this you could compare front to front the panels with MATCHED wilcoxon, as the samples were drawn from exactly the same population, but honnestly, after SI calculation you should already have made your mind and may not go on with statistics.

Thank you.

But it is not KaLwRij mice.

we need simple one

Not all patients with myeloma require a kidney biopsy. Myeloma kidney (cast nephropathy) is the most common cause of renal failure which requires a kidney biopsy to make a definitive diagnosis. However, myeloma patients often have associated hypercalcaemia with hypovolaemia. As a result, I always give a trial of isotonic fluids such as normal saline (up to 3-4 L/day) and assess response.

Clues to myeloma kidney include urine dipstick with a trace to 1+ proteinuria but with nephrotic-range proteinuria on UPCR or sulphosalisylic acid (SSA) together with a rising serum creatinine.

The second most common kidney disease is light chain deposition disease (LCDD). These cases behave like a rapidly progressive GN and will require biopsy.

At our institution, myeloma patients with myeloma kidney are managed palliatively (melphalan and dexamethasone only) since the overall prognosis is very poor.

We do at first Ficoll to obtain the MNCs but without Erythrocte lysis and then go for CD138+ beads for purification from Miltenyi. We do this manually with a magnet from Miltenyi and the respective column which is working as good as automated system, only requires some person to do it. The number of CD138+ cells strongly depends from the patient, often we get either nothing (everything below 50.000 cells is considered in our lab as nothing), or in between 100.000 and 200.000 cells from 20 ml of bone marrow. In rare cases we obtain more than 1 mio cells (up to approx. 10 mio cells was the maximum I got), this is strongly associated with disease progress. Try to get patients history and cases with disease progress for method establishment as this can only be successful when you have a proper amount of cells inside. And also do not hesitate to contact Miltenyi that they should come and help you, my experience in the past was that they did this and also gave free kit samples for testing purpose or borrowed even a magnet for 4 weeks for testing.

There are some reported studies showing evidence of some association between EBV and MM and specially so there could be a link to plasmablastic variant of myeloma

Yes. However you would have to do both the procedures- MLPA for del17p and RTPCR(cost effective than RQPCR) for the two translocations. MLPA has the advantage of covering many other deletions although their exact prognostic role is not well defined yet. To do both MLPA and RTPCR u need both DNA and RNA and I am not sure whether it will be cost and labour effective than FISH which is what u intend to achieve.

Dear Mayssa,

I just had a look at the publication you referred to. Unless you have the CDXX assays up and running, it will be impossible to identify which cells you have just by FSC/SSC. The fact that they showed up in a more or less defined group in a two parameter SSC/FSC plot is exceptional. It is,as far as I know, not possible to do the reverse. My suggestion would be to get the CDXXX assays up and running. Kind regards, Harrie Verhoeven.

Dear Mayassa,

FSC and SSC are not very useful signals for cellular properties. And they tend to be  highly dependent upon the type of instrument and its calibration/settings. Could  you give me more details about the instrument, light sources, detectors and the software you are using? Kind regards, Harrie Verhoeven.

Dear Neel

I assume you are referring to therapeutic ant-CD38 antibodies like daratumumab. It shouldn't be that difficult to get hold of a small aliquote from a local hospital that treats myeloma. Pharmacies discard any excess stuff. They will be happy to pass them on to you. Failing that, you have to go down the route suggested by Daniel. If you think your proposed experiments will produce results that will 'appeal' to the companies, they will be more than happy to support you. Yes, you will need a MTA.

Good luck

Siva

Our lab was able to ton transfect 8226, u266 and mm1s. They are extremely difficult to do, If you can use retrovirus, that is your best bet.

Otherwise, I would recommend including a fluorescent marker to enable FACS sorting a couple days following exposure. I would expect to perform multiple ficoll purifications if doing drug selection. If transient, Amaxa company has protocols already worked out with their electroporator. $$$$$ but pretty stunning results.  

We had mild success with cationic lipids. Keep the cells dense (but don't let over grow). DNA on the low end, transfection reagent in the mid range. If I get time I'll try to get you a specific protocol. If can't do virus try the amaxa systems

right they are now called lanza, you'll see your be cell protocols on their website.  http://www.lonza.com/products-services/bio-research/transfection/nucleofector-technology.aspx

Thank you for fast reply. I mean literature specific for kyphoplasty in multiple myeloma.

You can use a ligand-affinity resin from LigaTrap Technologies. It really works for human IgM purification from hybridoma or 10%FBS cell culture media..

Binding capacity 5-10mg/mL resin..

It can recover up to 90% IgM from the total load with > 95% purity.

Great question Raju:

Non-union of long bones are due to several reasons:

1.  Metastatic bone disease with enhance osteoclastic activities, includingnmyeloma; ant osteoclastic agents such a an intravenous bisphosphonates or denosumab have a potent effects in controlling such.

2.  Mal-alignment (a mechanical issues and may need surgical intervention

3. True non-union (e.g., atrumatic sub-trochanteric fractures (ASTFs) after prolonged use of bisphosphonates or denosumb.  These patents  likely to benefit with a short course (e.g., 3- 6 month) treatment with teriparatide injections (daily or EoD).  I refer to the following review article it the ResearchGate website (free to download) regarding this:

https://www.researchgate.net/publication/304029549_Optimum_duration_and_safety_of_long-term_use_of_potent_anti-resorptive_medications_in_osteoporosis?ev=prf_pub 

Best wishes,

Prof Sunil Wimalawansa

(suniljw@hotmail.com)

I find that IMDM+5% FBS + IL6  (we use 0.5ug/ml but this is probably overkill) works pretty well . Also, like Erna said, they like to be cozy, between 5E5 and 1E6/ml works pretty well. After they start expanding you can keep using bigger flasks, I'm routinely using T175 with 50E6 in 50 mls.  I have the feeling that each cell line has its own growth pattern though, so some trial and error will be necessary.

Cheers

Dominique

In the bone marrow the topographic distribution of plasma cells in cases of MM can be non homogeneous and in some cases "focal" aspects can be observed microscopically. The probability that one or more of these "hotbed" could constitute the prevalent material of a bone marrow aspiration must be considered. So, the case reported by Dr. Tacchi may be surprising for they entity but as much understandable and plausible

Hi Avadesh,

A number of things could significantly increase the risk of post-op infection in your patient; he is elderly, the treatment protocol he may be on presently (especially if dexamethasone or prednisolone is part of it) the disease (particularly if there are significant hypogammaglobulinaemia, and/or neutropaenia) and probably other co-morbidities (such as significant renal impairment). I will suggest your patient gets full (therapeutic) coverage of broad spectrum antibiotics for the procedure and the immediate post-op period, if any of the above (or indeed any other additional infection risk) is present, otherwise the prophylaxis could suffice.

All the best in your work.

John.

Hello,

In the latest update on MM treatment, Dr. Rajkumar discusses the combo of panobinostat with bortezomib and dexamethasone. 

You can read the article for free here. http://www.mayoclinicproceedings.org/article/S0025-6196(15)00895-2/abstract

I hope this helps.

Myeloma cells have a plasma cell or closely related phenotype. They are also committed to production of a particular immunoglobulin species based on Ig gene rearrangement. However, in some cases the anti-CD20 monoclonal rituximab will reduce paraprotein level despite the malignant cells in marrow being CD20 negative. There are others that know more about malignant plasma cells than I but I think there are reasons to believe that at least in some cases there is evidence for the malignant clone existing at least at the memory B cell stage. However, I am not aware of any evidence for clonal abnormality at earlier stages.

It may be that you are asking a rather complex question that has no one answer. Perhaps the question is at which stage of development does a mutation occur that predestines the cell to become a malignant clone. It is perhaps conceivable that this occurs at the pre or pro B cell stage or even at the stem cell stage but that the malignant clone still goes through maturation stages such as Ig gene rearrangement before expanding to take up bone marrow with a plasma cell phenotype. That would seem pretty unlikely and it might be that clear evidence is available to refute it but it might also be unknowable in practice for any given clone. What might be against it is that as far as I know we do not see malignant clones undergoing several different Ig gene rearrangements, which in theory should perhaps be the case if the cell acquired a malignant propensity at a pre B cell stage, or even at a later stage, considering that there is usually isotype switching to IgG.

The tranlocations responsible for the malignancy are known for a good proportion of myeloma clones. I suspect the people who have published on these would be able to give you a more black and white answer. Everything points to the critical mutation being at least at memory B cell stage I would say, but don't quote me on it.

In our Hospital,  the first therapy, for ineligible tranplanted patient is MPT.,exept renal  insufficienty.  If  patient have the renal insuffitiety, the  base therapy is  VD.

Best wishes

Little evidence of added efficacy at this point.

Would be surprising that 1-2 Bortezomib doses would add efficacy over many previous doses pre-transplant + high-dose melphalan.

Randomized trial ongoing at IFM.

Do you need a microarray to assay gene expression, exon expression, SNPs, or copy number variation? For gene expression there are quite a few suppliers available, such as Agilent and Affymetrix. Illumina also manufacture arrays. What I would recommend is speaking with the genomics core at your institution and asking them which arrays are compatible with their machines (unless you have one in your lab), and what their user base experience has been with the different types.

Dear Hareth: Thanks for this ver useful information. What is is the median OS and PFS in each case ? Median OS seems close to 3 years 

Would you be prepared to work with me on the data to derive estimates of QALYs? - or do you have these ?

Bw

Ifty

Back pain can be due to mechanical causes , which is common or due to inflammatory diseases or malignancy . The investigations are fine . In case , you are worried about calcium level , please check for Parathyroid hormone ( PTH ) level . His back pain seems to have improved & he is asymptomatic at present .

For CML, AML, ALL the best duration is 48 h culturing. We use cumulative method with FudR for 24h and 25 mkl Colcemid solution (concentration 10mkg/ml) in 10 ml culture for 4 h before harvesting. It's our unpublished technique. MM needs separation of plasmocytes.

Thank you so much dear Ewalds-Kvist

Sorry for writing this, but there is enough literature out there that gives you ideas. Just google cell separation.

Hi

I agree with Maulik, You should not go beyond 200 PCR product. ( Ideal range is 70-150, optimum 100). Now primer concentration, generally when I start working with new target I run a TEST run with 3 different concentration (200nm, 100nm,50nm). I have seen people use up to 5 uM of primer but if you go with higher concentration you may end with with Primer-primer dimer and that could mess up your Ct Values. As far as the concentration of other things, you can look up any kit available( I use Invitrogen) and follow their protocol and concentration you will be fine.

Dear Fernando,

here is the email address to  Mitch A. Phelps, who can give you an informed answer: mitch.phelps@osumc.edu.

Dear Avinash,

By checking the references to these papers you may find something useful:

Spectrochim Acta A Mol Biomol Spectrosc. 2014 Apr 5;123:241-8. doi: 10.1016/j.saa.2013.12.063. Competitive interactions of anti-carcinogens with serum albumin: a spectroscopic study of bendamustine and dexamethasone with the aid of chemometrics.

Wang Y, Zhu R, Ni Y, Kokot S.

Investigation of bendamustine HCL in a phase 2 study in women with resistant ovarian cancer. Baker AF, Roe DJ, Laughren C, Cohen JL, Wright HM, Clouser MC, Cui H, Alberts DS, Chambers SK. Invest New Drugs. 2013 Feb;31(1):160-6.

Bendamustine pharmacokinetic profile and exposure-response relationships in patients with indolent non-Hodgkin's lymphoma. Owen JS, Melhem M, Passarell JA, D'Andrea D, Darwish M, Kahl B. Cancer Chemother Pharmacol. 2010 Nov;66(6):1039-49

Competitive interactions of anti-carcinogens with serum albumin: a spectroscopic study of bendamustine and dexamethasone with the aid of chemometrics. Wang Y, Zhu R, Ni Y, Kokot S. Spectrochim Acta A Mol Biomol Spectrosc. 2014 Apr 5;123:241-8.

Bendamustine HCL for the treatment of relapsed indolent non-Hodgkin's lymphoma. Weide R. Ther Clin Risk Manag. 2008

You may want to look at publications by Tian E using a technique called TRI-FISH.

It is basically what Sabine mentioned a cytoplasmic Ig stain and then any other target of your liking.

We use it on a daily basis for patient sample here at the Myeloma Institute in Little Rock with excellent results.

It depends on how many MoAb may you use. In a minimal 5 colors panel we use a combination of CD19/CD38/CD138/CD45/CD56. Normal plasma cells use to be CD138/CD38/CD45/CD19 positive, CD56 negative, but abnormal plasma cells, as is multiple myeloma, are normally CD19/CD45 negative, and may express CD56. If you may incorporate a sixth color, we would use CD117, positive in more than 25% of MM plasma cells.

We have used multiple methods to introduce plasmids containting target cDNA or siRNA or shRNA into multiple myeloma cell lines. These methods include Amaxa Nucleofector, lipofectamine, and electroporation, and retrovirus vector expression system and Lentivirus vector expression system. The highest transfection (infection) efficiency among these methods is lentivirus vector expressing system, the gift from lab of Prof. Didier Trono (Université de Genève, Switzerland). Besides the high efficiency, cDNA in lentivirus expressing vector integrates with chromosome of the cells and thus prevents leaking of ectopic expressing genes in the vector from the expressed cells after culture of myeloma cells in vitro for long time.

(I assume that your treatment/stimulation/activation induces NFkB activation)

Extract proteins from treated and untreated cells. Perform a western blot using an antibody targeting phosphorylated NFkB. Stripe your membrane and stain again using an anti-NFkB antibody. Finally, check loading by actin staining.

NFkB bands should be similar, showing the constitutive expression, but you should see stronger pNFkB signal in the treated cells.

Cell Signalling has well working NFkB antibodies, as well as TNFa stimulated and resting cell lysates to be used as controls for the NFkB phosphorylation.

try 90% serum and 10% DMSO, believe me, it always works perfect. When you thaw it, try to thaw it quickly in 37 degree water bath. But when you freeze it down, try to freeze it slowly, for example, first put it in 4 degree, then -20, then -80, then liquid nitrogen. Or if you like just put it in strero box and leave it in -80, a couple of days later, relocate it in to liquid nitrogen. Good luck!

Mine RPMI8226 are semi adherent too. As longer you grow them you will have more and more adherent cells