NIH CLINICAL CENTER GRAND ROUNDS
Episode 2010-10
Time: 1:02:24
Recorded March 17, 2010

Therapeutic Gene Delivery Using Hematopoietic Stem Cells in Sickle Cell Disease
John F. Tisdale, MD
Senior Investigator, Molecular and Clinical Hematology Branch, NHLBI

Multiple Myeloma and Its Precursor (MGUS): Looking into the Future
Ola Landgren, MD, PhD
Investigator, Medical Oncology Branch, Center for Cancer Research, NCI

ANNOUNCER: Discussing Outstanding Science of the Past, Present and Future - this is NIH Clinical Center Grand Rounds.

(Music establishes, goes under VO)

ANNOUNCER: Greetings and welcome to NIH Clinical Center Grand Rounds, recorded March 17, 2010. On today's presentation, we'll hear from two scientists on diverse subjects. First, Dr. John Tisdale, senior investigator in the Molecular and Clinical Hematology Branch at the National Heart, Lung and Blood Institute, will speak about "Therapeutic Gene Delivery Using Hematopoietic Stem Cells in Sickle Cell Disease." He will be followed by Dr. Ola Landgren, an investigator with the Medical Oncology Branch in the Center for Cancer Research at the National Cancer Institute, who will speak on the topic "Multiple Myeloma and Its Precursor: Looking into the Future."

We take you to the Lipsett Ampitheater at the NIH Clinical Center in Bethesda, Maryland for today's presentation.

TISDALE: As far as learning objectives I want to first help us understand the role hematopoietic stem cells play in the potential curative treatment of sickle cell disease, update our recent clinical progress, using allogeneic hematopoietic stem cells for adult patients with severe sickle cell disease and finally review the progress of gene therapy using viruses to transgrow therapeutic genes to autologous stem cells for the treatment of sickle cell disease.

This slide reminds us it's been more than half a century since we described sickle cell anemia making it the first disease which the molecular defect was identified, resulting from a single substitution of the beda globin chain that polymerizes under conditions of low oxygen tension. This results in anemia, vasoclusive pain crises, severe end organ damage including stroke, renal failure and pulmonary hypertension and a markedly shortened life span of 40 years. In this abnormal hemoglobin among the red cell progeny of hematopoietic stem cells makes this ideal target disorder for hematopoietic stem cell based approach. There's been hype in the popular press regarding stem cells recently being able to cure diabetes, heart disease, Parkinson's and I want to try to give you a realistic up to date assessment where we are at least with hematopoietic stem cells for sickle cell disease.

So I'll be talking about adult derived hematopoietic stem cells, not the more controversial embryonic stem cells which have dominated the press of late. But recently Yamanaki and colleages have shown these cells are induced from mature cells such as skin through a combination of factors. These cells are now termed induced pluripotent stem cells, are very popular both for study in vitro and eventually for translation to the clinic.

The first proof of principal using IPS cells was shown in a sickle cell anemia mouse model and they induced it to pluripotency by exposure and corrected the sickle mutation, differentiated these cells into a hematopoietic progenitors, and then transplanted them back into the mice correcting the sickle cell phenotype.

This kind of work and others have really stimulated a great deal of interest in IPS cells and Francis Collins just announced last week the creation of an intramural national resource for IPS cells here on campus at NIH. It's really designed to translate this kind of work to the clinic.

So again, I'll be talking about hematopoietic stem cells. We're interested in these cells for a number of reason because they renew themselves and also to repopulate all of the blood elements.

So one can consider treatment of a number of disorders affecting these cells like severe combined immunodeficiency, affecting t-cells, chronic granuloma disease and granulocytes and the red cell progeny of hematopoietic stem cell if we could correct them could indeed correct sickle cell disease.

So we've taken a two prong approach to use stem cells as therapeutic vehicles. First, as allogeneic transplantation using bone marrow cells acquired from a donor that has the normal genotype. This currently requires an HLA matched sibling and patients have a 20% chance of having a match in their family.

The other approach is autologous stem cell quote gene therapy where we take the patients own bone marrow cells and transplant them after we corrected them by transferring a normal or therapeutic gene. This is required retroviral vectors that carry the gene because of their ability to permanently integrate into the chromosome of those stem cells. Hopefully we get reconstitution with normal red cells after either of these approaches.

We know that myloablative transplantation in children with sickle cell disease is curative. This is a report from 1996 from Mark Walter and colleagues. In an update recently with now over 200 children transplanted shows very high survival rates, more or less expected in an allo transplant setting for rejection. But graft versus host disease, a lethal complication of transplant persists in children at 15 to 20%. The more interesting observation that followed was that stable mixed chimers in some of these patients was sufficient to correct the disease phenotype. Stable mixed chimers means you have both cells of recipient and cells of donor contributing to hematopoiesis. So though they try to ablate the bone marrow to make the transplant work, 13 of 50 surviving patients had mixed chimers and as low as 11% donor cells in the white cell series in the periphery was associated with reversion of the phenotype of the sickle cell disease. We don't have a very high bar to reach to fix this disease. But this type of approach has not applied to adults because toxic conditions used to get patients ready and graft versus host disease really increases as we age. This is really limited application. In this disorder at least, to children.

So we and others sought to determine whether we could achieve intentional engraftment, get this stable state of mixed chimerism without ablation, without the associated side effects of the chemotherapy and radiation that's used to prepare these patients.

We had some clinical protocols that Rick Childs started some time ago that looked promising but appeared too toxic for application in sickle cell disease so went back to the lab to develop a mouse model of non-ablative transplant for sickle cell disease and specifically wanted to develop a regimen that would promote tolerance without the need for long-term immunosuppression and would allow for this holy grail of transplantation stable mixed chimerism.

So we used F-1 hybrid mice, a cross of black 6 that express KFB and KFD as donors and black 6 mice as recipients so this is a rejection prone model. This made studies easy because we could monitor the engraftment of white cells by flow cytometry, monitoring red cell series by looking at hemoglobins because they differ in these two strains.

Mice were mobilized to collect stem cells in the periphery, we wanted to mimic as close as possible the clinical situation. T-cells were collected at day 6 and infused to mice radiated with a relatively low dose of radiation, 300 centigrade.

Either a 30 day course of conventional immunosuppressive drugs cyclosporine, what was then a novel immunosuppressive drug rapamycin based on work from Jonathan Powell in Schwarz's group. At the end of 30 days immunosuppression was stopped and the mice were followed out to 33 weeks post transplantation.

So why rapamycin?

This is a cartoon of an immune response of t-cells encountering antigen complexes in the context of second signals liberate cytokine such as il-2 required for effector function and proliferation. Signal 1 in the absence of these signals renders t cells anergic and is required for tolerance. Cyclosporin is a great immunosuppressive drug but blocks activation through t-cells so theoretically could block intolerance if used in transplant model. Rapamycin blocks il-2 t-cell proliferation so it would allow the first signal to proceed without effector function of proliferation. Theoretically allowing the induction of tolerance.

Here I have shown engraftment in these mice over the first 33 weeks post transplantation in white cells, shown in the Y axis here this period of shading shows immunosuppression. And you can see cyclosporin treated mice has transient engraftment which they lost before the withdrawal of immunosuppression. Whereas the rapamycin treated mice reached levels around 80% which was stable beyond withdrawal of immunosuppression.

We applied the transplant regimen to a small cohort of sickle cell transgenic mice that express hemoglobin s shown here. This is a donor hemoglobin diffuse pattern and you can see all three recipient mice with hemoglobin coming from the donor strain, no more of the sickle hemoglobin.

We performed a control trial to evaluate safety and efficacy of peripheral blood stem cell mobilization in hetero zygotes, patients with sickle cell trait but otherwise normal. The kinetics were normal so you can see the cd34 cells mobilized in the periphery just as controls. Mild symptoms were more common but they had no crises like patients with sickle cell disease. And there was no jelling on thawing of these products. We wanted to cryopreserve the products so we could get a dose we knew that we had in hand before starting a preparative regimen for the patients. And there had been earlier literature suggesting problems with sickle cell trait blood during the freeze thaw.

We developed superior cryopreservation methods that are now in use in the department of transfusion medicine for actually all of our peripheral blood product. So in '03 we opened the protocol for non-ablative transplant for adult patients with sickle cell disease. Indications included severe end organ damage like stroke, pulmonary hypertension or renal insufficiency or a modifiable complication not ameliorated for the drug, hydroxyria, which was studied here early in the NIH clinical center. Conditioning was similar to that in the mouse 300 centigrade rapamycin and we added antibody to t-cells in order to further guard against development of graft versus host disease.

This table shows the ten patients for whom we have sufficient follow-up to comment on that accrued to this protocol. You can see their ages ranged from 16 to 45. It is an equal mix of male and female, most had hemoglobin FS. And the indications were predominantly vasoocclusive crises but included other indications such as stroke, renal damage, stress syndrome and a combination of many of the indications in several patients. Most had been managed by hydroxyrea with or without cell transfusion therapy. The patients tolerated conditioning really well which was really different than a standard transplant. No need for nutritional support common in conventional transplant, no acute or chronic graft versus host disease and no sickle cell anemia events, and nine of ten patients are with stable engraftment by donor.

This shows engraftment for the mouse earlier percent donor in the circulation. This time I divided between myeloid cells which are granulocytes and lymphoid cells which are t-cells and on the right hand Y axis is plotted hemoglobin. So you can see we had moderate levels of myeloid epigraftment that stabilized between 50 and 60% in this patient. More modest levels of lymphoid engraftment at 5, 10% settling at 15. This was associated with normalization of hemoglobin to 13-grams per deciliter.

This drop in the hemoglobin over the year was a period of therapeutic phlebotomy removing excess iron that had accumulated from transfusion therapy for stroke prevention in this patient so you can see it normalized back after we were able to decrease the iron back to a normal level.

This shows patients out to 30 months so you can see the pattern is similar as with the first patient. Engraftment rates of myeloid series of 80% and somewhere around 60% for T-cells. This mixed chimerism was sufficient in patients to correct the phenotype. You can see the hemoglobin before transplant after exchange at six months, one year an greater than a year. You can see they're normalized now in the 13 to 14 range. Also marked is a red cell homofluous which is a key feature of this disease, also normalized so reticulocyte count back to normal, total bilirubin back to normal and lDH back to normal.

We also saw reduction in the tricuspid regurgitant jet velocity in pulmonary hypertension which occurred immediately peritransplant and remained stable despite modest but significant increase in systolic blood pressure over the same time period.

One other satisfying feature is we weaned all patients on chronic narcotic therapy to completely off so this is plotting IV milligram of morphine equivalent per week in our patients at time of transplant. Around a thousand milligrams of IV morphine per week in these patients. And it took us quite some time to wean them off and especially right after discharge we had a spike in the usage because most patients had actually preferred to just stop cold turkey which is not possible when you have been on narcotics for this long. We got everybody off narcotics by around six months. This allogeneic transplant after low dose total body irradiation with rapamycin is sufficient to the sickle phenotype of 9 of 10 patients. This was associated with reversal of some of the end organ damage we see in this disease. And this low toxicity allowed us to apply this in adults with very severe disease. This mixed chimerism that we observed in the absence of graft versus host disease, really suggests at least operational tolerance. But we need longer follow-up and further accrual to validate these results.

As I mentioned earlier only about 20% of individuals have an HLA match within their family so we're still working on other ways that we can approach these patients including haplo transplantation which I don't have time to talk about today but also gene transfer to autologous sickle cells.

Working in Cindy Dunbar's lab a decade ago it was easy to cure anything in the mouse. High gene transfer rates were easily achieved, 50, 70% in circulation by simple methods using retroviral vectors. When these techniques were applied directly to human cells in vitro, equally high gene transfer rates were predicted by these in vitro assays. But when clinical trials started here at the NIH and elsewhere, in vivo levels of about 1 in 100,000 cells were seen, mostly these levels were transient. These were too low to expect clinical benefit.

This disconnect between the mouse and human in vivo and human in vivo made us go back and try to develop a better hematopoietic assay. When I was a post-doc in Cindy's lab we worked on non-human primate competitive repopulation model. This slide shows a decade of work in that model. The first thing that we did in Cindy's lab was try to determine whether a gene that we were putting into these hematopoietic cells was toxic to their differentiation or eliciting an immune responsech those two didn't seem to be limiting. We spent time worrying about what we did to the cells after we took them out because we take them out, select the very primitive progenitors and put them in culture for four days and expect them to behave like hematopoietic stem cells. These cells were differentiating so they went back to the animal or back into the human, not as stem cells any more but as progenitors. So we developed optimal cytokine support, developed clinically feasible methods for use in patients and then established by integration analysis true stem cell transduction.

We still had more work to do because for example in sickle cell disease we can't mobilize peripheral blood cells because of the toxicity of GCSF in this disease so we compared steady state bone marrow to the mobilized source of stem cells and found it was comparable in the model. Then we spent time worrying how much condition to give because if we lethally ablate before we put in the cells in the toxicity it will be much higher so we looked at lower doses of radiation in both mouse and non-human primate and busulfan which looked promising at low doses. So all this work predicted at the time that clinical success would be feasible at least in simple disorders where you might need 5% of an enzyme to correct the disease, then in fact turned out to be the case and this is the first demonstration in humans of efficacy of gene therapy approach where they showed correction of severe combined immunodeficiency using similar techniques in the human setting.

One of the problems that plagued the field is that these retroviral vectors when you put in the payload of beta glow bin recombine and become very unstable and it is hard to make high titer vectors. So this problem has consumed many post-doc lives until finally when one research group went to using HIV 1, lentiviral vectors based on HIV 1. This appeared promising as a means of transferring genes and he showed here some years ago hemoglobin synthesis in a mouse model in human beta thalasemia expressing human beta globin so we started a collaboration to develop this preclinically in the non-human primate.

We modified the vector because human and rhesus globin are similar. We produced vector at a pre-clinical scale using human immunodeficiency virus, had to develop all detection assays, optimize the transduction procedures. And initiated transplant studies in the monkeys to begin to bring this to the clinic.

And here I show in vivo expression of human beta globin in day 30 at transplantation in one animal with controls where human was mixed with rhesus at 0, 1, 5, 10 and 25%, and here you can see at day 30 around 5% human beta globin being produced in the animal after transplantation. When we followed a pair of animals we transplanted this way long term you can see the initial levels fell to levels which is at around 37 weeks became undetectable. About half a percent can be detected by this assay, below which you can't see. So the question became are these cells persisting and not expressing or are they going away because we didn't get into the stem cell compartment? And it turned out to be the latter. You can see if we look by DNA for integrated DNA among cells in circulation or cells in the bone marrow, in both animals the levels fell to .01% which is way lower than what would be therapeutically beneficial. It turns out that this is because of a species-specific block to transduction in the rhesus for human HIV one day vectors.

We looked at components of the innate immune system to try to circumvent this block and two of the main candidates we were studying were trim 5 alpha and apobec 3-g so we made viruses to get around this by combining portions of SIV in our plasmas we used to make virus. So we put simian capsid in the gag pal plasmid and simiman bif in the red virus and screened by titer to make virus.

So here is normal HIV, we can make 10 to the 7th particles very easily. If we add VIF we can make good titer virus. If we add capsid we can make good titer virus and if we add both we can still get respectful titer virus. So then we compared the viruses we could titer on human cell lines and rhesus cell lines to see if they were efficient at transducing these cells and you can see they all did well in human cell lines and human HIV performed poorly for rhesus cell lines. But the simbiant capsid transduced lymphocytes very well and human lymphocytes very well.

These are cells transduced with the standard HIV or the chimeric vector, these are rhesus cd34 cells transduced with standard HIV or chimeric vector and the chimeric vector outperforms the HIV-1 in all animals.

We then initiated competitive repopulation studies in a series of animals we took cd34 cells and split them with chimeric vector expressing green protein or standard HIV one base vector expressing yellow fluorescent protein, mixed them and transplanted them to see which would be better at transducing and reconstituting cell.

Here you can see in one animal the chimeric vector has far superior marking levels than HIV 1 with 30% versus 3% in granulosites an 20% in red cells versus a few percent for the standard HIV.

This is an animal where we put the entire product in and you can see 40 to 50% in the myeloid lymphoid cells and 20% in red cells. When we stain for specific surface markers like CD 3, CD 20 and CD 33, CD and CD 23, you can see 30 to 40% and among erythroid cells and platelets around 20%. So we were able to see high levels of marking among erythroid cells.

This is a peripheral smear of the second animal showing GFP among mature red cells in the circulation at a level expected to be of clinical benefit were we able to achieve with globin vectors. So I'll touch on where we're going from here.

Of course with the allogeneic stem cell transplantation protocol we need to continue accrual to make sure this holds up with the larger series of patients. But the big step is whether we can extend this to a haploid setting. As far as gene transfer we made efficient transduction in the animals. So now we're replacing the fluorescent marker genes with globin genes to compare vector designs to maximize expression of human globin. We're looking at additional safety measures, and I don't have time to talk about insertional mutagenesis. Maybe you can invite Dr. Dunbar on another grand rounds but looking for ways to diminish the complication in the trial I showed you, and finally we hope to be in clinical trials with this technique within the next several years so I'll stop here an acknowledge the crew and take questions.

[APPLAUSE]

We have time for questions.

QUESTION: How long are you keeping your sickle cell patients on siralimus?

TISDALE: This is a question we always get. So the original protocol was written so that we could wean siralamus when patients reached 100% limphoid chimerism and the graft was safe and they had no GVHD. No patient has gotten to 100% lymphoid chimeric yet but a couple of patients stopped their sirolomus on their own and one of whom always had levels all over the place. So I finally asked him besides last night when is the last time you have taken sirolomus?

He said six months ago which was the prior clinic appointment. Both of these patients that don't take medication have stable mixed chimerism as well so we feel we can based on the mouse model and experience with two patients begin to withdraw patients from sirolomus, so we have an amendment to the IRB to allow us to taper all patients after they have established stable mixed chimerism.

QUESTION: I am interested in the reversal of the end organ damage, I saw some of the patients have MRA, MAI abnormalities like stenosis. Did you have a chance to repeat the brain MRI to see if there was improvement? I know it's usually very difficult to reverse.

TISDALE: We have done a couple of MRIS long term follow-up in patient who did have abnormalities and so far it's only stable, no reversal.

LANDGREN: Thank you very much for inviting me here. So the title of my talk is multiple myeloma and its precursor looking into the future and the way I'll structure my talk is I'm going to give you a brief background from the literature and then I am going to shed light on activities and that we are doing and data we have published.

I have been with medical oncology since January last year so these first year has been a lot of prep work and I think 2010 will be the year when a lot of things are going to fall out clinically so we can open our studies.

So I don't have any conflicts of interest, nothing to disclose.

So the objective of my talk is going to be national history of myeloma precursor disease and novel approaches to treat multiple myeloma.

As a starting point, multiple myeloma and small ring myeloma are defined based on three criteria.

The first as you can see on the left is the level of monoclonal protein detected in blood.

The second is level of monoclonal plasma cells detected in the bone marrow and thirdly is presence or absence of end organ damage.

As you can see the cut off between small myeloma with regard to protein is three grams per decaliter. Above and below 10% of plasma in the bone marrow is the cut off between endosomal small ring. And end organ damage absence means endosomal small ring while presence would be myeloma and on the right corner of the slide you can see end organ damage, is the crab criteria, anemia, bone lesion, any of those would be a yes to end organ damage.

Myeloma is as you can see is really a clinical syndrome. We can do much better in the future.

Also as starting point I want to say multiple myeloma is classified as a rare disease in the literature but is the second most common hematologic malignancy, which affects about 20,000 Americans a year, twice as common in African Americans than whites.

Small ring myeloma, there's 5,000 cases diagnosed every year.

So what do we know about MGUS and small ring myelmoa progressing into myeloma? Most literature comes from rochester minnesota by Dr. Kyle and his group, they have shown they started this since 1960s that the risk of transformation, is about 1% per year and that risk seems to be constant over time. They have also shown this was published in 2007 in New England Journal, small ring myeloma patients have a much higher risk of transforming to full blown disease. That's the upward curve here so the first five years the risk is elevated and levels off and then beyond ten years it's quite parallel. Which suggests small ring myeloma is really an umbrella for a lot of heterogeneous cases. That's actually what they have shown in their own work as well from the Mayo Clinic that if one considers the level of bone marrow plasma cells in the bone marrow as well as the site of m spike taking these two clinical measures one can group patients into three groups.

And the group with both more than 10%, 3-gram it is group one, they have 87% of risk transforming into myeloma, the lowest group which is above three grams at the same time point is 39% so there's a twofold difference between the groups using the simple clinical measures.

They have also shown the addition of free light chains which can be now detected in blood is an independent predictor of progression and the Spanish study group have shown that using flow cytometry of the bone marrow one can look at the ratio between normal and abnormal plasma cells in the bone marrow and if the patient does more than 95% of normal plasma cells, that's an adverse factor.

They looked into the normal immunoglobulins and decrease, also an adverse factor and that's a reflection of the same thing if reflection of normal plasma cells are low, one expects immunoglobulins also to be low. At this very moment in 2010 in March we have two scales in the literature. The left is for the Mayo Clinic using the factors I mentioned.

About 10% plasma cells more than 3-gram protein and abnormal free light chain ratio. One, two, three risk factors and risk would go between 25 up to 76% at five years follow-up.

An based on the Spanish group the Spanish study group criteria, more than 95% of normal plasma cells and decreased immunoglobulins would be 0, 1, 2 points and the risk is 72% at five years. So this is as good as it gets in the literature.

I think we can do much better. So that's exactly what we're trying to do. We tried to focus on molecular profiling from precursor to full blown disease.

One of the questions we have addressed before I go into that and tell you about how we are trying to approach that here in the medical oncology branch, I want to show you the study just published about a year ago trying to answer the question if all myeloma patients actually are preceded by precursor states, as you all know when the patient has myeloma it's too late to address that question.

We wanted to see whether we could apply this question into some existing biobank here at the NIH. So using a study set up 10, 15 years ago, that allowed us to test that question. There were 77,000 healthy men and weapon, 55 to 74 years of age, every six years and followed prospectively for cancer so taking advantage of these fantastic resources we were able to identify total of 71 multiple myeloma patients and see there was an excess of males around seven years of age so they were quite representative compared to the myeloma in the population.

So using these unique resources we assayed for protein abnormalities at that time Mayo Clinic. This was carried out as a collaborative study by the NCI and the Mayo Clinic, Dr. Kyle's group. To summarize this study in two slides, on the left you can see number of years before multiple myeloma diagnoses, 2, 3, 4 up to 8 plus years. On the right you see the proportion of samples where we found evidence of abnormal protein. As you can see we found myeloma to be consistently preceded by MGUS.

This is an important finding. And reason I think it's important is because this is not a shocking result to me but it's nice to have it confirmed because now we can spend more time thinking about mechanisms of transformation and try to come up with strategies where we can target patients at high risk, try to look for patients with high risk and try to target them.

The second question to address was if the concentration of the h spike varied from early blood test up to the diagnosis of myeloma.

This is clinically an important question because the patients are typically followed every year by their physician and nobody really knows if the site of M spike goes up before onset of myeloma but that's the assumption that people have out there.

So we wanted to look at that. Again as you can see on the left, number of years before myeloma goes from 2 up to 8 plus years. Then you can see the median concentration. It's red on the right and we tested this statistically and it's highly significant P.025. However, one has to be careful when looking into this data because these are medians. If you look on an individual level you see a different pattern. There are some patients that have a stable m spike marked in green and there are those patients who go up gradually into myeloma.

If you summarize this individual slide into simplified slide, at least there seems to be two groups MGUS where the M spike gradually gets worse and worse and the patients eventually develops disease, myeloma and those that are non-evolving, stable, stable, stable and then bam, myeloma.

Why is this? I don't know.

Is it manifestation of genetic switch in tumor cell, a change in the host or is there a combination? This is something we're looking into right now. We have unpublished data. We have markers we look into normal plasma cells of these patients and we actually have seen that there are quite dramatic changes in the normal plasma cells, 10 plus years before onset of myeloma in some of these patients so that's something we're interested in.

So now what are we doing at the medical oncology branch on our clinical studies? We have three major focus areas, one is to focus on precursor disease and natural history studies to have a better understanding of molecular profiles between progressors versus non-progressers.

The second looks at high risk precursor disease, small ring myeloma, and see if we can come up with some treatment strategies.

And a third leg is to focus on people who have already developed multiple myeloma to develop targeted therapies.

This is work that we have been working on the past year so I don't have a lot of results but I have a lot of slides to show you about what you're doing and what we are going to set up.

So when it comes to the national history studies, in parallel with the clinical studies we use existing retrospective biobanks and this is an example of such a project. This is a study we're working in collaboration with the pathology lab Dr. Calvo and Merrik in collaboration with myeloma study group and the institute in Stockholm. So we have bone marrow biopsies in patients with MGUS followed for ten years, develop myeloma, biopsy of myeloma time point. And then we have biopsies of MGUS who were followed for the same period of time, 5 years who did not develop myeloma. We have samples here in the hospital and we are right now going to study tumor cell and bone microenvironmental cell changes using immunohistochemistry. As a next step we plan on use more molecularly focused technologies.

So this is a study with 20 MGUS in each of these two arms. As you can see this is a quite unique opportunity to do this. It would take a long, long time to set up this type of study prospectively so we're lucky having access to this retrospective biobank. Also we are in the process of starting another study with Dr. Calvo focusing on micrornas and their role of transformation. There's been a couple of studies in the literature suggesting that micrornas play a role in pathogenesis of multiple myeloma.

So in a similar fashion we are planning on approaching stored samples with MGUS that goes on to myeloma. As I mention we are already looking into the role of host immune markers using this retrospective resources.

So based on what we find in these studies we will identify candidate markers to plug into our prospect active study which I believe will open in a month or two. So this is the first prospective natural history study on myeloma in the country, which will be started here only 250 MGUS are going to be enrolled. We'll do a full work up with bone marrow blood an urine collection, going to follow these patients six, 12 months and then annually up for a five year interval. And do full work up at end of study as well if anyone develops into full blown myeloma. We're going to enroll 100 small ring patients with the same fashion, they of course will be seen more often.

The study has two components. One is the clinical component. So all the patients are undergoing immunohistochemistry.  This is an example of such a workup showing 15% plasma cells in the bone marrow and told you 10% cut-off so maybe you're thinking why does he show 50%, its not that much of a big deal, but what we also do is that we are looking into the bone marrow with flow cytometry.

On the left we have plasma cells. Done by Mary Ellis Stevenson. On these particular patients with 15% plasma cells, in the upper part in blue you see only two or three% of these plasmas are the normal. And in purple you have the normal plasma cells that are CD 45 negative and deemed to CD 19. So 97 to 98% of plasma cells in these patients are aberrant which is really bad. And on the left down you see Kappa Lamda paintings, positive with immunohistochemistry. So this is what we do for all patients.

We do immunohistochemistry, we do flow, we do work up with all blood markers as well as urine. Then we biobank samples and the plan in the study is to do gene expression profiling, array CGH and we will have material thinking of doing other things such as micrornas, et cetera, hopefully to do sequencing depending on post and development of technologies, et cetera. So I think this is going to be a very useful study to have a better understanding of molecular profiles of progressers versus non-progressers. Another thing we're interested in when it comes to transformation, is taking advantage of novel imaging technologies. Still in the textbook it says skeletal survey is the standard that's being done around the world including here to distinguish myeloma from non-myeloma when it comes to end organ damage, but as you know skeleton surveys are such an insensitive technology.

This is a patient I saw a couple of weeks ago. He has a diagnosis of small ring myeloma. He had a pet on the outside about a year ago and there was some suspicious lesion lab started to move so I felt it was reasonable to repeat and do a new FDG path. And as you can see, there are two lesions, one in the vertebra and one in the pelvis. 3.4 2.8 SUV, not strong but there. And if we did pet or MRI on small ring patients, I would think that maybe a quarter of them would have detectable lesions. These technologies are not super sensitive.

What we're doing is to develop new tracers so this is in collaboration with the molecular imaging group here in the hospital and with Pete Joyki et al. We're working on a protocol to use fluoride pet ct which based on the literature the pre-clinical literature, there are small studies indicating that this could be a very promising marker for early lesions.

Also there are new data indicating the dynamic contrast enhanced MRI could be used to visualize the androgenic switch in the bone marrow, I.e. microcirculation patterns from MGUS to myeloma. Data from Germany indicates differences between MGUS small ring and myeloma. We also want to do that.

We are writing a pilot study and depending on what we see here we'll plug into the perspective study as well.

What about early treatments strategies? So looking in the literature, these are the guidelines from the Mayo Clinic. It clearly says don't touch. Standard of care should be close follow-up every few months, repeat tests every two to three months and if stable, less often. Then it is consider clinical trials with aim to delay progression to symptomatic myeloma.

The problem is there are not a lot of studies, and in fact, no open clinical trials in the United States for small ring patients.

This is a summary of the world literature of published studies for asymptomatic myeloma. The biggest study published so far is the third one from the bottom from Arkansas, Dr. Barlogi published in blood, 76 smoldering patients, treated with 200-milligrams of thalidomide, and because of the toxicity, 86% had dose limitations down to 50% and more than 50% of patients had to go off study due to side effects, so that study had a lot of problems.

There is a study reported in New Orleans three months ago, this is the only to my knowledge ongoing study in the world as of right now.

This is a randomized phase 3 study led by the Spanish collaborative group, they have plans to enroll 60 patients in each arm. And they're basically using multiple myeloma therapy that's exactly what they do. So on the left side they have the 60 patients who go into the treatment arm 25-milligrams we are day 1 through 21, day 1 to 4 and 12 to 15. They give nine cycles of this therapy. For those who don't deal with this drug, this is myeloma therapy. A lot of patients, they go in complete remission after four cycles or maybe three cycles or five cycles or up to 8. So given nine cycles to asymptomatic patients, that's potent therapy. Then they go down to the maintenance dose which is ten milligrams daily, day 1 to 21 every second month. And on the right side, they have the follow-up that was kind of funny, Spain is catholic country, they call it therapeutic abstention. So the primary end point of the study is time to progression to symptomatic disease. And the second end point is response rate duration of response progression free survival, overall survival safety and tolerability.

So they reported this at ash and had a median follow-up of 14 months. They don't have data to answer all these questions. They had a response, 91% overall response rate for intention to treat dose patients receiving nine cycles but they had a lot of toxicity, as one would expect.

So what we are thinking is there something we could do that does not include conventional myeloma therapy? To say those patients who regress so we don't use all the ammunition up front. So how can we think about this? If we don't do anything we know what's going to happen. They're going to progress and proliferate.

So we are thinking two scenarios. One is the dream scenario and I don't think we are there yet but the dream scenario is if you have this wonderful drug you treat the patient early, the patient is now cured, there is no myeloma there. The other scenario is probably the more realistic scenario I think and that's when you treat and you get rid of a lot of disease but not all disease you need to repeat it so you convert myeloma into a chronic disease, and myeloma is like hypertension.

So one thing we're looking into now is actually using natural kelo cells as part of the innate immune system of the patients.

Natural kelo cells have been recognized several years but only a few years back people have understood a little bit more about regulation of these cells. They go after cells infected by infection and they go after tumor cells but unfortunately when it comes to tumor cells they use MHC 1 kill ligands to down regulate activity. In myeloma that's exactly what happens that the tumor cells are being recognized but then they inactivate the natural kelo cells. We know from allogeneic transplant when there's a mismatch between donor and recipient, this does not happen. Because then there is no match for the receptors. A lot of the anti-myeloma effect in allogeneic transplant is because NK cells are activated.

We are right now looking into the opportunity to use a monoclonal antibody, that's an antibody developed by a French company.  Which conceptually mimics the mismatch situation because it blocks the receptor. So this is something we're looking into and we are in the process of developing a clinical trial for small ring patients. We also are looking into other approaches as well. This is just an example.

Lastly I wanted to shed some light on what we're doing for myloma. As of right now we have made a strategic decision to basically attack myeloma on the flanks. So we go early on, we go for pre-cursor disease, try to develop strategies for early treatment and to understand the natural history. The other flank is people who relapse and refractory disease.

We are developing targeted therapies there so for myeloma in the literature people talk about the so-called novel drugs. They have been around ten years but even if nothing happen 40 years, it's quite novel so they include thaldomyde. None of these drugs have been designed because they target particular targets. So working together with others, we are now looking into using the MEK ERK pathway.

Those tumor cells with MAF translocation also depend on MAC signaling and are about 20% so adding the numbers together there's a little overlap, takes you to 50%. About 50% of the myeloma patients presumably should be susceptible to MEK inhibition.

Also the MEK ERK pathway is activated in osteoclast for their differentiation and when they differentiate they secrete cytokines that the myeloma cells like so studies are indicating blocking the macro pathway in osteoclast would have an anti-myeloma affect by simply hitting the osteoclast in a microenvironment.

So right now we're opening a study in a month or so using this drug, this is an Astra Zeneca drug, CTEP approved by the IRB so hopefully open soon.

This is my last slide. Working with the National Institutes of Craniofacial and Skeletal Disease, Dental Institute. Dr. Pamela Robe and Dr. Mike Keel and others, we are right now working on this project trying to develop mouse models so Dr. Robe's group have developed a model where they are able to grow human bone under the skin of a nod mouse. So we have been implanting bone marrow stromal cells with the carrier used for orthopedic surgery.

Some of these cells, they have capability to give rise to bones after eight weeks there is growth of human bone under the skin. There is a brief background. At this time there's two other myeloma models out there. One is developed by Josh Epstein in Arkansas where he has taken aborted fetuses bone and put them in mice and that's a very complicated model. He has developed that model further and is using rabbit bone so that's that model. Then there is another model using mice that have been manipulated to develop mouse myeloma.

So studying mouse myeloma in mice or studying with these fetal or rabbit bones, these are the two models. So what we have been doing here under supervision of Dr. Keel is to inject, this is where we are now, luciferase expressing myeloma cell lines. As you can see on the slide we see growth after three weeks of injection of these cell lines.

This is something we're very interested in and the goal is to take primary cells as a next step and we are excited because we hope to be able to use this for drug development as well as trying to dig deeper into pathogenesis.

I would like to thank my collaborators and also all my extramural colleagues.

Thank you very much.

[APPLAUSE]

QUESTION: [INAUDIBLE]

LANDGREN: The question is for the biobanking how we're going to handle the cells. We do conventional biopsy for diagnostic purposes and then we do aspirates of the bone marrow as well. That goes a little bit goes to the pathology department for diagnostics, a little goes to the flow lab for flow cytometry and the other component goes for sorting. And then we keep the CD 138 positive and negative and the supernatant being alloquated.

ANNOUNCER: You've been listening to NIH Clinical Center Grand Rounds recorded March 17, 2010. On today's presentation, we heard from two scientists on diverse subjects. First, Dr. John Tisdale, senior investigator in the Molecular and Clinical Hematology Branch at the National Heart, Lung and Blood Institute, talked about "Therapeutic Gene Delivery Using Hematopoietic Stem Cells in Sickle Cell Disease. He was followed by Dr. Ola Landgren, an investigator with the Medical Oncology Branch in the Center for Cancer Research at the National Cancer Institute, who spoke on the topic "Multiple Myeloma and Its Precursor: Looking into the Future." You can see a closed-captioned videocast of this lecture by logging onto http://videocast.nih.gov -- click the "Past Events" link -- or by clicking the "View Videocast" link on the podcast homepage at www.cc.nih.gov/podcast. The NIH CLINICAL CENTER GRAND ROUNDS podcast is a presentation of the NIH Clinical Center, Office of Communications, Patient Recruitment and Public Liaison. For more information about clinical research going on every day at the NIH Clinical Center, log on to http://clinicalcenter.nih.gov. From America's Clinical Research Hospital, this has been NIH CLINICAL CENTER GRAND ROUNDS. In Bethesda, Maryland, I'm Bill Schmalfeldt at the National Institutes of Health, an agency of the United States Department of Health and Human Services.