Science topic

Multiple Myeloma - Science topic

Multiple Myeloma is a malignancy of mature PLASMA CELLS engaging in monoclonal immunoglobulin production. It is characterized by hyperglobulinemia, excess Bence-Jones proteins (free monoclonal IMMUNOGLOBULIN LIGHT CHAINS) in the urine, skeletal destruction, bone pain, and fractures. Other features include ANEMIA; HYPERCALCEMIA; and RENAL INSUFFICIENCY.
Questions related to Multiple Myeloma
  • asked a question related to Multiple Myeloma
Question
3 answers
I am currently using the Quanti-Nova SYBR Green PCR Kit from Qiagen and I would like to know how much cDNA (ng) will be sufficient for amplification of my genes of interest (BMP2, 6 and SMAD6). Does anyone have worked with this kit previously?
And another question if at a qPCR experiment we use the same amount and some of the samples are not amplified, this means that they are undetected or I must modify the concentration so that qPCR can detect the very low concentration for the sake of statistical analysis?
Relevant answer
Answer
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!
  • asked a question related to Multiple Myeloma
Question
29 answers
At present there are several radiological methods and radioisotope available, but what is the most effective in detecting bone disease in multiple myeloma?
Relevant answer
Answer
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.”
  • asked a question related to Multiple Myeloma
Question
3 answers
Hello, I am using the EZNA total RNA kit I for the extraction of RNA from patients with multiple myeloma (bone marrow). In three different experiments, I consistently extract RNA AND gDNA, and I am not sure where the problem is, in the homogenization process (I use the 21 gauge syringe, they also suggest the homogenization mini column), maybe the silica membrane clots, is the quantity of cells too much so the gDNA cannot be removed effieciently? Do I need to perform a DNAase I pepsis?
Thank you in advance.
Relevant answer
Answer
Dnase I from Qiagen.
  • asked a question related to Multiple Myeloma
Question
4 answers
I collect samples for patients with multiple myeloma, with many questions about the nature of their lifestyle, I found big differences such as smoking, where they live and the nature of food, but there is one common factor that I found by chance, which is excessive tantrums that remain for not a little time. My question is, is it possible that this is the main effect of the abnormal production of plasma cells from the bone marrow?
Relevant answer
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.
  • asked a question related to Multiple Myeloma
Question
6 answers
Hi, I am a beginner in the field of cancer genomics. I am reading gene expression profiling papers in which researchers classify the cancer samples into two groups based on expression of group of genes. for example "High group" "Low group" and do survival analysis, then they associate these groups with other molecular and clinical parameters for example serum B2M levels, serum creatinine levels for 17p del, trisomy of 3. Some researchers classify the cancer samples into 10 groups. Now if I am proposing a cancer classification schemes and presenting a survival model based on 2 groups or 10 groups, How should I assess the predictive power of my proposed classification model and simultaneously how do i compare predictive power of mine with other survival models? Thanks you in advance.
Relevant answer
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
  • asked a question related to Multiple Myeloma
Question
1 answer
A 58 years old male reports to you with a fissure like ulcer on the elevated part of lingual ridge from the past 2 months. he further tells two episodes of whitish flakes coming out from the fissure. his medical records show that he is under medication for multiple myeloma for the past 8 months.
what is your likely diagnosis and how would you manage the case?
Relevant answer
Answer
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
  • asked a question related to Multiple Myeloma
Question
4 answers
Which cell line offers the highest transfection efficiency? in Multiple myeloma or Neuroblastoma. I heard in multiple myeloma RPMI-8226 and U266 is hard to transfect.
and Neuroblastoma IMR-32 is low transfection efficiency. Is there other cell line to easily transfection? in multiple myeloma or neuroblastoma.
Relevant answer
Answer
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...
  • asked a question related to Multiple Myeloma
Question
3 answers
In my group we are planning to use an ELISA kit in order to measure VEGF in serum from relapse multiple myeloma patients. We wonder whether we could have problems with the peak in immunoglobulin we expect at that point in our patients, in term of interference with the measurement of VEGF. Does anyone use this kind of kit in this sort of patient?
Thank you in advance
  • asked a question related to Multiple Myeloma
Question
6 answers
Dear Researchers,
I am wondering if anyone can help and tell me about
Which type of COVID-19 vaccine is safe to use it for a patient with
Multiple myeloma (MM), (cancer of plasma cells)
(Multiple myeloma
also known as plasma cell myeloma and simply myeloma, is a cancer of plasma cells, a type of white blood cell that normally produces antibodies.)
I need to know this information urgently for one of my relatives.
Thanks in advance.
Best wishes,
Zainab
Relevant answer
Answer
Good question. We have to wait the results of the research work in the future.
  • asked a question related to Multiple Myeloma
Question
5 answers
I am culturing U266, a suspension cell line and run out of the untreated flask. Can I culture the suspension cells in treated flask?
Relevant answer
Answer
It will depend on the future study that you have to conduct with your cells.
  • asked a question related to Multiple Myeloma
Question
1 answer
Hi!
I have been looking for publications where CD38 knock-out (or even knock-down) in multiple myeloma cell lines has been generated, but I have difficulty finding them.
Have you come across such publications or were you able to produce a CD38 knock-out in multiple myeloma cell lines such as RPMI8226 or MM.1S? Which method did you use?
Thank you!
Relevant answer
Answer
Hello!
I find some publications about knock down CD38 in multiple myeloma
Maybe this articles will help for you?
  • asked a question related to Multiple Myeloma
Question
3 answers
Can anyone share some insights about:
1. expansion of T cells using feeder cells, like a protocol. My goal is to expand T cells from patient with multiple myeloma.
2. is it better to use feeder cells than CD3/CD28 cocktail to activate T cells or use both of them?
Thanks
Relevant answer
Answer
Thanks Lael Werner and Laurence Stuart Dawkins-Hall very much for your helps.
  • asked a question related to Multiple Myeloma
Question
4 answers
I am working on culturing of human multiple myeloma plasma cells (Primary cells) and it shows very slow growth with the use of DMEM with 10% FBS, 1% streptomycin-penicillium 2mM L-Glutamine, other RPMI-1640.). But it shows very slow growth rate. Please kind to enough provide the detailed of culture conditions of human multiple myeloma cells.
Relevant answer
Answer
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.
  • asked a question related to Multiple Myeloma
Question
1 answer
I have got a promoter region which is size about 1000bp from human genome. I'm trying to find cis acting elements corresponds for gene regulation in multiple myeloma. What are the freely available methods for this?
Relevant answer
Answer
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).
  • asked a question related to Multiple Myeloma
Question
2 answers
I am trying to get an insight on whether to use athymic nude mice or NOD-scid IL2Rg null (NOG) mice for my RPMI8226 multiple myeloma (MM) intratibial bone implantation experiments. There are references for intratibial implantation for both of these mice backgrounds. With athymic nude mice, these mice are a lot less expensive than the NOG and have also displayed the MM characteristics that people expect. However, the more recent papers for intratibial tumor models have been using NOG with no mention of any athymic nude mice disadvantages if any and why they would be less advantageous. The tumor-take percentages for nude mice are also excellent. Thank you for your advice. Vinay ~
Clin Cancer Res. 2009 Mar 15;15(6):1998-2009
J Bone Miner Res. 2009 Jul;24(7):1150-61
PLoS One. 2013;8(2):e57641
Relevant answer
Answer
Frank Arguello Thank you so much
  • asked a question related to Multiple Myeloma
Question
2 answers
Currently, I am using RPMI 8226 and want to culture this cell on polystyrene substrate. I have to coat polystyrene substrate with fibronectin to ensure better cell attachment. I am wondering what is the optimum concentration of fibronectin that I should use. Also, if you can let me know what type of fibronectin I should use or if there is any other product type that I can use, I will really appreciate it.
Relevant answer
Answer
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.
  • asked a question related to Multiple Myeloma
Question
14 answers
Dear Colleagues,
My Dad (66 years old) has recently beed recently diagnosed with multiple myeloma. Apart from significantly elevated monoclonal protein, he has no symptoms. He has had his blood protein levels high for the past 5 years. Detailed medical exams showed that his bones, kidneys, and blood work (other than what I have already mentioned) are all fine. My Dad has always been fit and he is still feeling well overall.
He has an opportunity to join a clinical study that uses daratumumab combined with standard treatment for multiple myeloma. My understanding is that the treatment he would get is considered chemotherapy.
Is undergoing the chemotherapy the right thing to do now, considering multiple side effects of it (with or without daratumumab)? Or should he wait until symptoms begin to appear. My Dad’s doctors do not seem to share the same opinion on this matter.
I would deeply appreciate suggestions. Please note that I am a neurolignuist, not an MD. Therefore, I may have worded something incorrectly here. However, I will gladly clarify or provide more detailed information if helpful.
Thank you very much in advance,
Monika
Relevant answer
Answer
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:
  • asked a question related to Multiple Myeloma
Question
2 answers
I would like to isolate Leukopaks 3-5 billion cells/ml from Multiple Myeloma patient.. and how EGF will help to improve the patient Leukopaks..? without EGF is it possible to isolate 3-5 billion cells/ml
Relevant answer
Answer
Yes..
  • asked a question related to Multiple Myeloma
Question
5 answers
Hi,
I am trying to transfect multiple myeloma cell lines with siRNAs using the Amaxa nucleofector. I extract RNA with the TRIzol method 48h after the nucleofection, and I find that while 260/280 ratios are OK, the concentration (50-200 ng/uL) and 260/230 ratios are low (0,6-1) except for the negative control, which looks better (700 ng/ul, 260/280 1.8 and 260/230 1.6). I usually do a ethanol precipitation as a last step and then I let RNAs dry about 30min before I add warm H2O DEPC. I don't know if the low ratio is due to the fact that my cells are dying because of the nucleofection (I find that 50% cells are dead 48h after nucleofection) or because I am not evaporating ethanol correctly.
Should I try to evaporate ethanol better? Or should I try to transfect by other means? Will I see a reduction in mRNA by qPCR extracting RNA after 24h?
Thank you so much,
Ane
Relevant answer
Answer
Yes, you should evaporate ethanol completely. Any trace amount of ethanol could affect the quality of RNA.
Good luck!
  • asked a question related to Multiple Myeloma
Question
3 answers
I have purchased 100mg of melphalan powder for in vitro study in multiple myeloma cells. Anyone know what is the best solvent for melphalan, which is less toxic to the cells? How long is the stability of melphalan in solvent? Thank you.
Relevant answer
Answer
Will try DMSO, thank you Saganuwan Alhaji Saganuwan Samir G Pandya
  • asked a question related to Multiple Myeloma
Question
6 answers
I am about to isolate CD138+ plasma cells using a Miltenyi Biotec MACS kit from the PBMCs of healthy donors.
Although I know that circulating plasma cells are scarce in normal individuals, I need to isolate them to use as control for primary malignant plasma cells that I am working on.
So, I need to know if I can successfully isolate plasma cells from peripheral blood of healthy individuals and the possible yields.
Thank you in advance!
Relevant answer
Answer
In normal person detection of plasma cell as an reactive cell especially in infective or inflammatory process is possible
  • asked a question related to Multiple Myeloma
Question
3 answers
I wonder if randomized and controlled studies exist for the following triplet therapies in Multiple Myeloma:
1) "Daratumumab, pomalidomide and dexamethasone"
2) "Carfilzomib, pomalidomide, and dexamethasone"
Regards,
Ronny
Relevant answer
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;
  • asked a question related to Multiple Myeloma
Question
3 answers
We are currently having a hard time getting TAMs (especially Multiple Myeloma associated macrophages) from peripheral blood monocytes. Do you have any suggestions or possible protocols for doing so?
Relevant answer
Answer
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
  • asked a question related to Multiple Myeloma
Question
3 answers
I am doing a research project where I am comparing CD38 and VS38 for the detection of plasma cells in multiple myeloma cases.
For each sample I did two plasma cell panels: 1) CD38, CD138, CD19, CD45, CD3 and 2) VS38, CD138, CD19, CD45, CD3. Both CD38 and VS38 were conjugated with FITC. I should point out that with the cytoplasmic VS38 I have lost a significant number of cells compared to CD38.
Is it appropriate to use MFI to compare the two antibodies given that the experiment conditions were the same for the two panels? What other data should I use to compare and analyse the two antibodies?
Any help or feedback is greatly appreciated.
Relevant answer
Answer
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.
  • asked a question related to Multiple Myeloma
Question
4 answers
I am afraid of that our KaLwRij stain has been Genetically contaminated with C57BL/6 or other stain.
Because 5TGM1-GFP tumor do not easy to be induced in some mice from same parents.
Relevant answer
Answer
Thank you.
But it is not KaLwRij mice.
  • asked a question related to Multiple Myeloma
Question
5 answers
Good day for all
I'm wondering if here is any new biochemical parameters for evaluation of bone disease in multiple myeloma patients in comparison with imaging studies
I wish I can hear any suggestion form experts colleagues
regards
Relevant answer
Answer
we need simple one
  • asked a question related to Multiple Myeloma
Question
3 answers
AKI in patients with multiple myeloma. Role of kidney biopsy in the management
Relevant answer
Answer
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.
  • asked a question related to Multiple Myeloma
Question
18 answers
First, I'm using Ficoll Histopaque centrifugation to obtain mononuclear cells. Then, I'm doing RBC lysis. I observe a drastic loss of CD138+ cells after that (as checked using FACS and two separate validated CD138 antibodies). E.g. a sample that contains 18% plasmocytes, has practically no plasmocytes left after Histopaque isolation.
I want to use MACS CD138+ Microbeads but they fail since the Histpaque-isolated cells are devoid of plasmocytes.
Does anyone have such problems? What would you recommend? I've read about autoMACS Pro being good for such cases, but I don't have access to this device.
Relevant answer
Answer
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.
  • asked a question related to Multiple Myeloma
Question
2 answers
Any comments or suggestions  in reference to EBV role in myeloma please
Relevant answer
Answer
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
  • asked a question related to Multiple Myeloma
Question
1 answer
The International Myeloma Working Group proposed a risk classification for multiple myeloma based on FISH. For high risk is necessary at least one of the following: del17p, t(4;14), or t(14;16).
As FISH is expensive, I am thinking in other alternatives to study this 3 cytogenetic abnormalities (CA).
Relevant answer
Answer
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.
  • asked a question related to Multiple Myeloma
Question
5 answers
Je ne peux pas connaître la gate de cette lignée dans le nuage des point de la figure fsc/ssc de la cytométrie en flux 
Relevant answer
Answer
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.
  • asked a question related to Multiple Myeloma
Question
3 answers
I want to know how to gate the population of this cell line (U266)
Relevant answer
Answer
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.
  • asked a question related to Multiple Myeloma
Question
2 answers
I am looking to obtain a few antibodies that are FDA approved or in clinical testing for multiple myeloma. The antibodies are sold by Jansen and Sanofi.
Who would I reach out to see if I can get this antibody for (non-patient) research use?
Relevant answer
Answer
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
  • asked a question related to Multiple Myeloma
Question
4 answers
Dear All,
Does anyone has a positive experience with MM1.S or/and U266 multiple myeloma cells transfection. I need to transfect these cells and not to electroporate and now looking for a reagent that can give more then 30% cells transfected. I appreciate any suggestion.
Elena
Relevant answer
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
  • asked a question related to Multiple Myeloma
Question
5 answers
Is there any indication for kyphoplaty in multiple myeloma who have vertebral involvement. And if there is any indication how many verebrae you can do per sstion?
Relevant answer
Thank you for fast reply. I mean literature specific for kyphoplasty in multiple myeloma.
  • asked a question related to Multiple Myeloma
Question
5 answers
Dear All,
I'm looking for an efficient protocol to purify secreted IgM (Human) from cell culture.
Thank you so much
Selena
Relevant answer
Answer
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.
  • asked a question related to Multiple Myeloma
Question
5 answers
Presenting a follow up of 18 months of a 40 years male after multiple myeloma, where we could achieve union after intramedullary nailing and chemotherapy.
Relevant answer
Answer
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:
Best wishes,
Prof Sunil Wimalawansa
  • asked a question related to Multiple Myeloma
Question
16 answers
I am currently working on multiple myeloma cell lines. The problem I am experiencing is that these cells are no longer functional after 3 weeks in culture. They are still viable but not functional and respond differently to the treatment. Is it very normal with RPMI 8226, U266, OPM2 cell lines or not?
Relevant answer
Answer
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
  • asked a question related to Multiple Myeloma
Question
6 answers
I have a patient that was diagnosed with Multiple myeloma, around 95% plasma cells in the bone marrow. In the flow citometry though, clonal plasma cells are just 10%. any explanation for this?
the Moab used were CD45/38/138/19/20/117/56/cyLamda/cyKappa
Relevant answer
Answer
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
  • asked a question related to Multiple Myeloma
Question
3 answers
Elderly patient on Chemotherapy for multiple myeloma is on Anti microbial prophylaxis, does the incidence of post op endophthalmitis increase?
what are the precautions?
Relevant answer
Answer
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.
  • asked a question related to Multiple Myeloma
Question
1 answer
We have a few patients who have either relapsed following velcade and revlimid or progressed during these treatments. We have access to panabinostat but not to carfilzomib or daratumumab. I would appreciate your views
Relevant answer
Hello,
In the latest update on MM treatment, Dr. Rajkumar discusses the combo of panobinostat with bortezomib and dexamethasone. 
I hope this helps.
  • asked a question related to Multiple Myeloma
Question
2 answers
In one recent study (Agirre et al 2015, Genome Research) the authors suggest B cell enhancers are inactivated in multiple myeloma. In stem cells/progenitors cells the aforementioned B cell enhancers are inactive and become activated in B cells. Is it known for certain that cancer cells in MM come from transformation of B cells or is it possible that they come from defective stem/progenitor cells?
Relevant answer
Answer
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.
  • asked a question related to Multiple Myeloma
Question
9 answers
There are many regimens developed for the treatment of multiple myeloma. A subset of patient is in frail and some combination therapies are often difficult to apply. Because treatment strategy in our institution is not concreted, i'm seeking good treatment strategy which can apply to patients with myeloma, regardless of their comorbidity or physical status.
Note) in Japan (i'm Japanese), P-I study has completed in the Cy+Bor+Dex, Vel+Mel+PSL, and Vel+Thal+Dex regimens, but dose reduction was generally required due to higher toxicity than results of foreign study.
Currently, following agents are available.
 [First-line]
 Chemothrapeuic agents (cyclophosphamide, melphalan, doxorubicin)
 bortezomib
 lenalidomide
 corticosteroids, such as dexamethasone or prednisolone
 [Refractory or relapsed]
 panovinostat
 thalidomide
 [Following agents will be available near the future]
 carfilzomib
 elotuzumab
Please let me know your consideration!
example)
1st: Bor+Dex
2nd: Bor+Dex+Thal (if poorly responded)
= treatment-free (or maintenance with Bor+Dex?)=
3rd: Bor+Len+Dex (when CRAB appeared or not responded)
4th: Bor+panovinostat+Dex
5th: Len+Dex+elotuzumab or carfilzomib-based regimen (when available).
Relevant answer
Answer
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
  • asked a question related to Multiple Myeloma
Question
3 answers
There was a phase-2 trial by Roussel et.al in Blood 2010
[Bortezomib and high-dose melphalan as conditioning regimen before autologous stem cell transplantation in patients with de novo multiple myeloma: a phase 2 study of the Intergroupe Francophone du Myelome (IFM) ]
which showed CR was higher if velcade was added. But I couldn't find any data on long term outcomes. Is there any data on this? 
Relevant answer
Answer
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.
  • asked a question related to Multiple Myeloma
Question
3 answers
(I expect to have around 100 samples)
Relevant answer
Answer
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.
  • asked a question related to Multiple Myeloma
Question
6 answers
im looking for data on the use of lenalidomide in Multiple Myeloma patients in actual clinical practice. If the data is audit data thats fine too. Im happy to work with anyone for publication on this (im a medical statistician) 
thanks
ifty 
Relevant answer
Answer
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
  • asked a question related to Multiple Myeloma
Question
9 answers
The patient has Hypothyroidism, Hypertension, Type 2 Diabetes, & Vitiligo. 
He had an episode of SIADH 2 months ago. 
Can someone help interpret his investigations?
His CBC is as follows:
White Blood Cells 10.15x103/uL
Red Blood Cells 4.34x106/uL
Hemoglobin 13.1 g/dL
Hematocrit 40.9 %
MCV 94.2 fL
MCH 30.1 pg
MCHC 31.9 g/dL
RDW 14.6 %
Platelets 349x103/DL
MPV 9.5 fL
Neutrophils 72.7 %
Lymphocytes 19.7 % 
Monocytes 4.3 %
Eosinophils 0.5 %
Basophils 1.1 %
Large Unstained Cells 1.6 % 
Neutrophils Abs 7.38x103/uL
Lymphocytes Abs 2.00x103/uL 
Monocytes Abs 0.44x103/uL
Eosinophils Abs 0.06x103/uL 
Basophils Abs 0.11x103uL
LUC Abs 0.16 103/uL [*] 0.00 0.50
His Chemistry
Glucose 129 mg/dL
Phosphorus 3.4 mg/dL
Uric Acid 7.7 mg/dL
Creatinine Serum 0.95 mg/dL
Urea 22.1 mg/dL
Total Protein 7.68 g/dL
Albumin 4.70 g/dL 
Globulin 2.98 g/dL
SGOT(AST) 28 U/L 
SGPT(ALT) 33 U/L
Gamma GT 14 U/L
Alkaline Phosphatase 85 U/L
LDH 356 U/L 
Bilirubin Total 0.56 mg/dL
CPK 61 U/L
Calcium 10.56 mg/dL 
Sodium 140 mmol/L
Potassium 5.0 mmol/L 
Chloride 100 mmol/L 
His Lipid Profile:
Cholesterol Total 173 mg/dL
Triglycerides 120 mg/dL 
HDL Cholesterol 53.9 mg/dL 
LDL (Calculated) 95 mg/dL 
Relevant answer
Answer
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 .
  • asked a question related to Multiple Myeloma
Question
3 answers
Does anyone know which culture duration is ideal for cytogenetics diagnosis of  each bone marrow malignancy? (CML, ALL,AML, MDS, Multiple Myeloma). Which cell type do I need to grow in each case? Any personal knowledge or citation would be appreciated.
Relevant answer
Answer
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.
  • asked a question related to Multiple Myeloma
Question
2 answers
social risk factors of MM are my major concerns instead of biological. Please public health expert are needed here to guide.
Relevant answer
Answer
Thank you so much dear Ewalds-Kvist
  • asked a question related to Multiple Myeloma
Question
8 answers
Please let me know of methods & protocols required for the isolation of pure individual subsets of lymphocytes like B,T,NK & Plasma Cells from lymphocytes of blood. Isolation of Lymphocytes from blood is easy but how to further isolate its subtypes. Please suggest some protocols for that. Thank You.
Relevant answer
Answer
Sorry for writing this, but there is enough literature out there that gives you ideas. Just google cell separation.
  • asked a question related to Multiple Myeloma
Question
6 answers
I'm working on some members of B7 family protein transcripts on multiple myeloma cells and I'm about to standardize my RT-PCR protocol, but actually I don't know how to start the process. Theorical Tm of my oligos are 50 to 65°C and the length of the products are 100 to 600 bp. I'm using Promega GoTaq Flexi DNA polymerase and dNTPs at 100uM. The cDNA is synthesized with Promega MMLV-RT and RNA is extracted using single step method. Should I start adjusting concentrations first? What concentrations should I use for oligos and overall master mix?
Hope I'm clear and thank you. Greetings!
Relevant answer
Answer
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.
  • asked a question related to Multiple Myeloma
Question
2 answers
Adenosine deaminase (adenosine aminohydrolase, EC 3.5.4.4; ADA) ) is a enzyme ubiquitous, widely distributed in human tissues, and common in blood peripheral cells, like lymphocytes, granulocytes and erythrocytes. Serum ADA levels were raised in patients with haematological malignancies.
Relevant answer
Dear Fernando,
here is the email address to  Mitch A. Phelps, who can give you an informed answer: mitch.phelps@osumc.edu.
  • asked a question related to Multiple Myeloma
Question
2 answers
I am following a method using phosphate buffer, but unable to find out any degradation of the bendamustine HCl even after 6h.
Relevant answer
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
  • asked a question related to Multiple Myeloma
Question
6 answers
We found that all our myeloma samples were negative when we observed 13qS319, 13qS34, p53, t(11:14), t(4:14) and t(14:16) with FISH. Then we tried to sort CD138+ MM cells with FacS Aria III and afterward applied to the FISH. However, we saw that we lost our cells too much.
Could anyone suggest about our methodology or any other alternative methods?
Relevant answer
Answer
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.
  • asked a question related to Multiple Myeloma
Question
3 answers
Immunophenotyping.
Relevant answer
Answer
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.
  • asked a question related to Multiple Myeloma
Question
12 answers
I am wondering if there is an effective transfection protocol, that increases the efficiency in this suspension cells. Any tips are welcome.
Relevant answer
Answer
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.
  • asked a question related to Multiple Myeloma
Question
2 answers
I want to determine the constitutive NF-kappaB in MM cell lines by western or/and RT-PCR but I don't know the criteria. Are negative/positive control cell lines needed to see which is constitutively activated?
Relevant answer
Answer
(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.
  • asked a question related to Multiple Myeloma
Question
5 answers
We would like to freeze primary myeloma cells before testing the compounds. Does anyone have any experience with this? What cryomedia did you use? Any advice on thawing the cells?
Relevant answer
Answer
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!
  • asked a question related to Multiple Myeloma
Question
9 answers
Does anyone have any experience in culturing the RPMI8226 myeloma cell line? I am using T25 from BD falcon, and what I am observing is a significant fraction of cells are adhering to the plastic and forming colonies rather than growing in suspension.
Relevant answer
Answer
Mine RPMI8226 are semi adherent too. As longer you grow them you will have more and more adherent cells