from Leadership Medica n. 273/2009

A meeting with three experts from Milan's Ospedale Maggiore Policlinico:

Prof. Glorgio lambertenghi, head of the bone marrow transplantation centre; dr. Francesco bertoli, of the transplant immunology and transfusion centre; and dr. Davide soligo, hematologist at the bone marrow transplantation centre

Neoplasms still represent one of the main, causes of death throughout the world, among them, leukemia, lymphomas and other hematological diseases are of particular interest because of their high incidence and our advanced level of understanding of some of their pathogenetic mechanisms.
However, the success rate of bone marrow transplantation - still regarded as the treatment of choice in these diseases - is strictly related to the possibility of identifying the genetic factors which may lead to the rejection of the transplanted marrow itself; consequently, it is still considered preferable to use family HLA-compatible donors.
The serological typing of class I and II HLA antigens has been partially superseded by molecular (or genomic) investigations, making it possible to reduce the incidence of rejection and Graft Versus Host Disease (GVHD). As long ago as the early 1950s, Professor Emil J. Freireich was one of the researchers who attempted to give a clearer definition of leukemia after the discovery of the role of transferrin in the transport of serum iron. In his pioneering studies of 1958, he compared two different polychemotherapy schedules:

1. daily doses of methotrexafe and 6-mercapfopurine
2. intermittent doses (every three days) of the same drugs.

The National Cancer Institute made further discoveries during the 1960s by observing the natural history of acute leukemia and by studying the effects of platelet transfusion and chemotherapy in acute leukemia patients. Since then, research has never stopped. Professor Robert Peter Gale is another scientist who has dedicated himself to leukemia research, particularly the potentially curative effects of bone marrow transplantation, a technique which has now become the therapy of choice in the treatment of acute lymphoblastic, as well as acute and chronic myeloid leukemia.
His studies revealed the role of T-lymphocytes and the importance of supportive care in reducing the incidence of interstitial pneumonia and other clinical complications. In addition to leukemia, bone marrow transplantation is also considered to be potentially curative of other autoimmune diseases, such as lupus erythematosus and rheumatoid arthritis. In recent years,  Leadership Medica has published articles by both Emil Freireich of the University of Houston's Department of Medicine: No. 1/91) and Robert Peter Gale (of the Division of Oncology and Hematology at UCLA's School of Medicine: No. 10/90); we have also dedicated an entire issue to  the subject of bone marrow transplantation, publishing articles c/es by experts in various medical fields (No. 3/91).
We would now like to give an update on the current state of research in this area and verify the results which have been achieved since the beginning of the '90s

In the Leadership Medica editorial office meeting among the Editor in Chief, Genina Iacobone,
prof. Giorgio Lambertenghi Deliliers, Dr. Davide Soligo, and Dr. Francesco Bertolini

Interview with prof. Giorgio Lambertenghi Deliliers, Head of the Bone Marrow Transplantation Centre, Ospedale Maggiore Policlinico, Milan

BONE MARROW TRANSPLANTATION: SIGNIFICANT IMPROVEMENTS

Prof. Lambertenghi, what is the future of bone marrow transplantation?
The future lies in separating stem cells from the umbilical cord or peripheral blood making it no longer necessary to use bone marrow. These cells would be cultured in vitro grown and then transplanted. Consequently, if will soon no longer be necessary to work on bone marrow: it will be enough to transplant even a limited number of stem cells expanded in vitro by means of hemopoietic growth factors.

What is the current objective?
The current objective is to find alternative sources of stem cells for patients who do not have an available donor in their family, a situation which affects something like 70% of all of patients requiring bone marrow transplantation. At the moment, these have three possibilities open to them. The first is to find an HLA-compatible donor in the Bone Marrow Donors' Registry; and here it is worth pointing out the importance of international cooperation because the bigger the Registry, the greater the chances of finding a suitable donor. The second could be to use cord blood (which would otherwise he eliminated in any case) in order to build up a cord blood bank which could offer a source of supply of stem cells other than bone marrow. The third possibility is an autologous transplantation of peripheral blood stem cells, which seem to have a number of advantages over bone marrow cells. Stem cells can also be cultured and grown in vitro.

Have there been any changes in the criteria used for selecting transplantation candidates over the last few years?
Allogeneic bone marrow transplantation has become the treatment of choice for various malignant blood diseases which cannot be treated (or rather cured) by means of traditional chemotherapy, for bone marrow aplasia and some immuno-deficiency syndromes particularly characteristic of infancy. There is no doubt that, for these diseases which cannot be cured by traditional therapy, bone marrow transplantation is the treatment of choice. We are speaking here of allogeneic bone marrow transplantation from an HLA identical family donor. However, the probability of finding such a donor is only about 30%, which means that 70% of patients do not have a family donor available. Given that their diseases cannot be cured using traditional therapies, an alternative has to be found for these patients, and so it is necessary to find other sources of hemopoietic stem cells. As I have said, there are three possible ways of doing this: the International Registry of unrelated donors, where patients can find an HLA identical donor; the Cord Blood Bank, which is in the process of being created; the use of stem cells cultured and expanded in vitro. This final option is thought to be the most promising for the future.

The harvest bone marrow from donors under total anesthesia has often acted as a psychological and material deterrent. Has anything changed in this technique?
In reality little has changed, although I can say that the procedure now takes much less time. Today, it only takes about 30 minutes to an hour to draw a litre or one and a half litre of bone marrow. Nevertheless, it looks like new methods will shortly replace the technique of bone marrow aspiration under general anesthesia.

What is the state of the art concerning the technical complications of transplantation?
In Allogeneic transplantation from a family donor, the complications have been considerably reduced. This is particularly the case with GVh which can now be very well controlled using immunosuppressant drugs and pretransplantation procedures such as T-lymphocyte depletion.
Infectious complications can now be prevented and treated in a highly specific way using antibiotics, antimycotics and environmental prevention measures (such as laminar flow sterile rooms). However, there are still a number of complications with allogeneic bone marrow transplantations from unrelated donors.
A lot of research still has to be done concerning this type of transplantation because grade II, III and IV (which is often fatal) GVt-ID continues to exist in up to 80% of patients. Infectious complications due to cytomegalovirus are still to be feared, and there is also graft failure - which is usually rare in allogeneic bone marrow transplantation from related donors, but occurs in 20-30% of patients in whom bone marrow from non-related donors is used.

What can be done in this area?
I have already mentioned the registry of unrelated donors, but this is related to two other fundamental considerations. The first is the fact that, given the considerable polymorphism or the HLA system, close international cooperation is necessary if we are to have a numerically large registry, because the probability of finding a perfectly identical donor is between 1/50,000 and 1/100,000.As far as typing is concerned, we now have some very refined  molecular and  biological techniques for determining the identity of the HLA system.
We will talk later about the collection of stem cells from the umbilical cord and peripheral blood, and the in vitro expansion of stem cells.

Given the new HLA typing methods, how have the criteria for selecting donors changed?

In the past, the HLA characterization of patients was done using serological methods, and errors were made in 20-30% of patients, particularly in determining second class HLA antigens.
As a result, donors and recipients were sometimes thought to be identical when they were not.
The reason for these errors is that these serological methods were not very sensitive. Today, HLA - DR second class antigens are studied using molecular biology methods to analyse the patients' DNA.
The genes coding HLA have been sequenced, and we now have molecular probes available for the various segments of the HLA gene. Here, I am referring to a molecular biology method which can dissect HLA very precisely, and which is the one used in today's best Centres. It is a technique which is essentially based on the use of oligonucleotides (that is, small fragments of complementary DNA and PCR to amplify DNA sequences.
The technique is called PCR-SSO: Polymerase Chain Reaction Sequence Specific Oligotyping. We can now obtain much more precise information about the donors, avoid many of the errors of the past and discarding donors who appear to be identical but are really not.
Using these techniques, we will be able to reduce the differences between donors and recipients, and therefore ensure that transplants from registry donors have a much lower incidence of GVHD, which still greatly affects the quality of life and post-transplant mortality. The largest experience of patients transplanted from the registry is that of the Feed Hutchinson Center in Seattle, and they have compared a group of past patients who were typed using serological with a group in which the identify of HLA was determined at the molecular level.
The survival curves of the patients with better HiA-DR loci identification were clearly better. All of this explains why, over the last two years, there has been a lot of insistence on the need to determine HLA-DR using high resolution molecular biology methods.

Interview with dr. Francesco Bertolini, of the Transplant Immunology and Transfusion Centre, Ospedale Maggiore Policlinico, Milan

TRANSPLANTATION OF CORD BLOOD: A FUTURE PATH

One of the directions chosen for the future is the cord blood. Can you describe the relevant characteristics of this method?
The official starting point was in 1989 when, at the beginning of the year, the group headed by Hal Broxmeyer in Indianapolis published a very important paper showing the possibility of using cord blood collected immediately after birth as a source of stem cells for use in transplants. In a period of only a few months, what was originally only a hypothesis was made reality by Prof. Gluckman, who performed the first transplantation in a 7 year old boy with chronic anemia using cord blood collected at the birth of his brother (who was not affected by the disease and was identical in terms of compatibility antigens. The first transplant was so successful that now, 5 years later, the patient is very well and can be considered clinically cured. This result was published, and since 1989, there have been about 40 cases of patients who have received blood obtained from the umbilical cord at the time of the birth of a compatible sibling. The data comes from a very heterogeneous series of patients, both in terms of disease (out of 35 cases, there are at least /different diagnoses) and in terms of age (from 1 to 16 years). The most interesting data concern the fake in the vast majority of cases and the fact that GVHD reactions were mild or absent. Although there are clearly too few cases to be able to draw any definitive conclusions, there is an increasing interest and some projects of cord blood banking are arising, above all because the typed cord blood is immediately available for transplantation. A source of this kind has a number of advantages over bone marrow donor registries, because the time between making a request for a compatible donor from the International Registry, identifying the donor and actually carrying gut the transplantation may be several months. At best, if takes at least 6 months, which means that the condition of some patients become worse. Frozen cord blood can be quickly transported to any country in the world. It's not difficult to understand that it can travel far more easily than a donor. Another relevant point is that, if we analyse the type of donors in the Registry, the vast majority are of Caucasian origin and there are no compatible donors for numerous ethnic minorities which are not adequately represented in the Registry. By systematically collecting cord blood, it is possible to balance the donors of different races and obtain cells available for transplantation even for ethnic minorities. In the United States, for example, at least 30% of ethnic minorities are not represented in the Registry, and this creates serious complications. Another advantage is that, as we are dealing with blood from subjects who have not had an extrauterine life, the possibility that the cells are contaminated with a virus is extremely low. The example of cytomegalovirus, which is particularly dangerous to the recipient, is very informative: in large European and American cities, 70% of the donors are carriers of this virus, against less than 1% of newborns. The final advantage is that cord blood cells are also a very useful vector for future gene therapies. The program is currently under way to set up world cord blood banks are based on two experiences: the American New York Bank Center, with more than 2000 samples that have already allowed two transplants, and the European collaborative effort of countries such as France, Great Britain, Germany and Italy, which has so far led to the collection of about 1000 units. More than 300 units have been collected in Milan and they are HLA-typed and ready for a possible transplant. These countries are now organizing themselves in order to be able to dialogue with the United States Registry. I believe that, in about two years time, it will be possible for patients who need a bone marrow transplant to make a request to both the Adult Donor and the Blood Cell Registry.

Can you describe the scientific aspects of this methodology and its main phases?
The collection technique is very simple and does not involve any risk to either mother or child. This is confirmed by more than 3000 samples taken in the USA and Europe. At the time of birth, the umbilical cord is clamped as normal and the residual blood remaining in the placenta is collected in special bags containing anticoagulants by means of a sterile technique. This blood is extremely rich in stem cells, and can either be used as if is or be separated to reduce the volume to be frozen. At present, cord blood collected in this way is frozen in its entirety and preserved in liquid nitrogen at -196° C, in this way, it can be kept available for possible transplantation for decades.

How many stem cells are collected using this technique?
About 70 ml of cord blood are collected, although depends very much on the baby's weight of birth. The concentration of stem cells is the same, if not more the number in bone marrow. An further interesting point that these cells are much more immunologically “naïve” bone marrow cells, insofar or they come from a subject who has never encountered bacteria, viruses and other antigens during his or her intrauterine life, except those of the mother. This absence of previous stimulation is probably the reason for the cells' reduced aggression and the lower incidence of GVHD.

This method, requires collaboration of gynecologists. Do you have this collaboration?
Making an effective collection is only possible if there is good collaboration between the physicians responsible for it, the gynecologists and the obstetric staff. Our experience during this first year of setting up the bank is that the level of collaboration has been extremely high; however, it is necessary to continue to sensitize not only gynecologists, but also the physicians who care for patients with blood diseases,  given that we should collect blood from the umbilical cord of the brother. This must become an immediately available option for relatives a fleeted by these types of disease, and if is an opportunity which must not be lost.

In the United States, there are proposals to make compulsory the collodion of umbilical cord blood, a substance which is still considered a waste product in hospitals. From a technical point of view, what difficulties need to be overcome for this method to become routine?
The main technical difficulty is how to preserve the cord blood of all newborn babies. At present, it is absolutely impossible because of the technical difficulties involved in setting up banks of such proportions.

If transport were available from the gynecology and obstetrics centres to a cord blood bank, would there be sufficient technical time for the drawing, collecting, freezing and transport of the blood?
Certainly. The main problem would be economic. The deposit of cord blood in a bank involves typing tests which, as we have seen, are complex; tests for blood-transmitted diseases, such as HIV, the various types of hepatitis, etc.; and the cryopreservation of the material. The cost of all this is something like one million lire per sample, which is acceptable but cannot be met by the limited funds currently available for research.
Prof. Lambertenghi would like to point out that, in Italy, a bone marrow sample costs about 10 million lire.
Have there been cases in which a pregnancy has been planned in order to collect cord blood for transplantation? Some families have proven to be very open to this possibility. In the past months we have collected cord blood from 12 brothers of children with blood diseases. In two cases, the samples were also compatible and we are preparing for the transplantation.

Let us now consider a method which is even more projected into the future: the use of stem cells from peripheral blood.
Professor Lambertenghi, can you explain the main aspects of this?
We know that hemopoietic stem cells are present in small numbers in bone marrow and fetal blood and, for some time, it has been known that they are also present in the peripheral blood of normal subjects, although in a smaller percentage. We also know that stem cells are regulated by a large number of growth factors, which have recently been carefully characterized functionally and molecularly, most of which molecules. I would like add one point concerning cord blood stem cells. These cells are usually present in very low numbers in normal healthy people, but it has been found that their number increases considerable after about 15 days in patients treated with a short cycle of chemotherapy. For example, when a patient receives a chemotherapeutic agent (usually cyclophosphamide), this is followed by a phase of bone marrow aplasia and, after about 15 days, there is a peak of stem cells in the peripheral blood. At this point, there is the possibility of collecting these stem cells by leukapheresis (that is, machines which collect leukocytes). It has been observed that the leukocytes collected by leukapheresis are rich in stem cells and their use in transplantation has been hypothesised. At this stage, if has been surprisingly confirmed that this type of stem cell takes very well, but produces a faster recovery of leukocyte and platelet count than is the case in patients transplanted with bone marrow.
These techniques have been subsequently further improved, and it has been found that, the haemopoietic cell growth factor (in particular, G-CSF or GM-CSFj is added to the short cycle of chemotherapy, there is an even greater increase in the number of peripheral blood stem cells.
For example, the transplantation of stem cells obtained from peripheral blood has been performed in subjects with breast carcinoma whose bone marrow was absolutely undamaged by disease; patients, that is, to whom if is licit to administer chemotherapy with growth factors.
The sequence is this: polychemotherapy is followed by the administration of growth factor, and the stem cells are then harvested and frozen; the patient next receives supramaximal doses of radiotherapy in an attempt to eradicate the disease; finally, the patient is reinjected with the peripheral blood stem cells.
A final consideration: it is no longer even necessary to administer chemotherapy and then the growth factor; in normal subjects, itis enough to administer a growth factor such as G-CSF for five days and there is a very large increase in peripheral stem cells. It should also be said that treatment with G-CSF is not toxic, given that its side effects are minimal. In brief: a normal subject undergoes treatment with growth factor, the peripheral blood cells are collected, and these are then transplanted info other HLA identical subjects.

Interview with Dr. Davide Soligo, Hematologist at the Bone Marrow Transplantation Centre, Ospedale Maggiore Policlinico, Milan

STEM CELLS CULTURED IN VITRO; THE LAST FRONTIER OF BONE MARROW TRANSPLANTATION SCIENCE

The in vitro cultivation of stem cells is in the fore front of bone marrow transplantation research. Can you describe the current situation?
Stem cells are self-renewing cells, which are present in small numbers, proliferate slowly and are responsible for the maintenance and production of all blood cells.

We now know most of the factors regulating the proliferation and differentiation of stem cells.
These cells can be isolated from bone marrow, peripheral blood or from the umbilical cord, and cultured in vitro in simple cell culture flasks or in the recently developed "bioreactors", in which he growth factors and all the other nutrients are inserted in a continuous cycle. The stem cells reproduce and increase in number.

Does this overcome the problem of compatibility?
Compatibility is a fundamental problem, and remains as such. However, what is more important is that the donor is less involved than when giving bone marrow.
Secondly, by expanding the number of stem cells, we can overcome the problem of rejection or lack of "take" often found in some patients probably because the number of reinfused stem cells is insufficient: in vitro expansion can overcome this problem. Another point to consider is the expansion of the umbilical cord cells, which have so far been used for transplantations in babies and small children, the biggest being a 50 kg boy.
What we are afraid of is that there are too few umbilical cord stem cells for transplantation in adults. However, we do know that umbilical cord stem cells have considerable growth potential, probably because they are less mature and therefore more easily stimulated. Given that they have this potential to expand very much, if is therefore easy to imagine that the transplantation of umbilical cord stem cells in adults could become an optimal choice in the future.

What is the numerical increase in stem cells in vivo with G-CSF and over what period of time?
Briefly, one hundred times in five days. This increase occurs 5-6 days after the administration of the growth factor alone; but when using sequential chemotherapy and growth factor, it is necessary to wait much longer.

How long do you think it will take before this transplantation technique is widely used?
I believe that if will be applied technically in 2-3 years and that, in five years, the transplantation of peripheral blood stem cells (autologously, using an HLA-identical donor an unrelated donor) will almost completely replace classical bone marrow transplantation. About three years ago, a growth factor for hemopoietic cells called "stem cell factor" (SCF) was identified which, in vitro, enormously stimulates the proliferation of stem cells. In vivo clinical trials are currently underway using this new growth factor in combination with either G-CSF GN-CSF.
These combinations appear to induce an even greater increase in the number of peripheral blood stem cells, from an increase of 100 times to one of 1000 times.

Genina Iacobone
Direttore