Authors:

Sophie Fessl

Boris Labar

Steven Pavletic

Abstract

Haematopoietic stem cell transplantation (HSCT) is a routine treatment for many haematologic malignant and non-malignant diseases. This article gives an overview of key points in the 60-year history of HSCT programs in Europe. It also discusses the close relationship between Zagreb transplant team and European School of Oncology in Milan (ESO). This model is an example of how ESO has played a critical role in the advancement of modern clinical medicine.

Introduction

Haematopoietic stem cell transplantation (HSCT) is an effective treatment for acute leukaemia, severe aplastic anaemia, and many hereditary haematological disorders. Bone marrow and peripheral blood cells are the main source of haematopoietic stem cells. In recent years, the transplantation outcome has significantly improved. The availability of the donor no longer poses a problem, as transplantation has been performed routinely from related, unrelated, and haploidentical donors. A high success rate has been reported in elderly patients transplanted with reduced-intensity conditioning. Improved patient care has decreased toxicity and mortality after treatment. However, it took since the 1950s to get to this stage. Although the crunch point, the first successful modern allogeneic bone-marrow transplant (BMT) to cure leukaemia, was performed in the US by E Donnall Thomas, significant contributing work – both before and after Thomas’ success – stems from Europe.

History - Some Key Events

The impetus behind much of the work in the field of BMT originated from concerns arising after the dropping of atomic bombs on Hiroshima and Nagasaki in 1945 and the subsequent Cold War-era paranoia. In the nascent nuclear age, radiation sickness emerged as a new and substantial threat. The pressing need for a cure became evident, particularly for what came to be known as the bone marrow syndrome, resulting from excessive radiation exposure. The United States military played an active role in supporting research aimed at finding a solution to radiation sickness ^[1].

In 1949, Jacobson and colleagues ^[2] made a pivotal discovery when they found that shielding mice spleens with lead provided protection from total body irradiation (TBI). Two years later, Lorenz and colleagues ^[3] reported the radiation protection of mice and guinea pigs by infusing marrow cells. Initially, many investigators, including Jacobson, believed that the radiation protection stemmed from some humoral factor(s) in the spleen or marrow. However, by the mid-1950s, this "humoral hypothesis" was firmly rejected. Several laboratories convincingly demonstrated that the radiation protection was attributed to the seeding of the marrow by donor cells.

This discovery was met with enthusiasm due to its implications for cell biology and the treatment of patients with life-threatening blood disorders. The principle of HSCT was straightforward: high-dose radiation/chemotherapy would both eradicate the diseased marrow and suppress the patient's immune cells, allowing for the acceptance of a donor graft. Within one year of the crucial rodent studies, Thomas ^[4] and colleagues demonstrated that marrow could be safely infused into leukaemia patients, leading to engraftment, even though ultimately, leukaemia relapsed.

In 1958, Mathé's group ^[5] attempted to rescue six nuclear reactor workers, from Vinca Yugoslavia, accidentally exposed to TBI through marrow transplantation. Four of the six survived, although donor cells persisted only transiently. In 1965, Mathé and colleagues ^[6] treated a leukaemia patient with TBI and marrows from six relatives, without knowledge of histocompatibility. A brother's marrow engrafted, and the patient entered remission but eventually succumbed to a complication, graft-versus-host disease (GVHD). Building on early observations by Barnes and Loutit ^[7] in mice, Mathé [https://www.oncopedia.wiki/key-players/georges-mathe] ^[8] coined the term "graft-vs.-leukemia effect."

These transplants were conducted without a comprehensive understanding of conditioning regimens, histocompatibility matching, and the control of graft-versus-host disease (GVHD). They were directly based on research involving inbred mice, where histocompatibility matching is not an absolute necessity. In 1967, van Bekkum and de Vries ^[9] asserted, "These failures have occurred mainly because the clinical applications were undertaken too soon, most of them before even the minimum basic knowledge required to bridge the gap between mouse and patient had been obtained." Clinical HSCT was deemed a total failure, and prominent immunologists declared that the barrier between individuals could never be crossed.

In 1958, van Rood ^[10] and colleagues made a significant discovery: during pregnancy, approximately one-third of women developed antibodies against human leukocyte antigens (HLA). This revelation provided a pathway to unravel the genetics of HLA. Subsequent studies delved into the role of these antigens in HSCT, contributing to a heightened understanding of the importance of HLA typing and, consequently, improving donor selection strategies.

In 1968, van Bekkum, Balner and colleagues successfully formulated an HSCT protocol in monkeys, sharing this information not only in the Netherlands but also with Good and co-workers in the United States. Within the same year, three patients—two in the United States and one in the Netherlands, all suffering from a congenital immune deficiency - underwent successful transplantation with hematopoietic stem cells from an HLA-identical sibling donor. The first successful HLA-matched allogeneic bone marrow transplant was reported by Good in Minnesota for a pediatric patient with severe combined immunodeficiency syndrome. ^[11]

In March 1969, E Donnall Thomas and his team carried out the first transplant using a matched sibling donor for a patient with advanced leukaemia. ^[12]

The patient suffered a blast crisis of chronic myelogenous leukaemia (CML), his sister donor was HLA matched. Although the transplantation was successful and the leukaemia went into remission, the patient died 56 days later of pneumonia caused by cytomegalovirus.

ED Thomas stated during the Nobel price ceremony: ^[13]

These studies marked the beginning of the “modern” era of human allogeneic marrow grafting. … In summary, marrow grafting has progressed from a highly experimental procedure to being accepted as the preferred form of treatment for a wide variety of diseases at many varying stages of disease. Subsequent years witnessed an expanding capability among various centres to perform allogeneic HSCT successfully in patients with hematologic malignancies, including acute leukaemia.

In an interview conducted by one of the authors (S.F.) Steven Pavletic refers to a stand-still in Europe: “Seattle [where E. D. Thomas worked] took over in the late 1960s, and this is the time when there was no European clinical activity in bone marrow transplant.” In the 1970s, Pavletic describes activities picking up again in Europe, particularly in the Netherlands. Bruno Speck ^[14] helped found the bone marrow transplantation program in Leiden (together with Jon van Rood). At Leiden, Bruno Speck also became interested in observations by George Mathe [https://www.oncopedia.wiki/key-players/georges-mathe]: some patients with aplastic anaemia given antilymphocyte globulin (ATG) and bone marrow infusions improved, even when bone marrow engraftment was transient or not even evident. Over the next decade (the 1970s – Speck returned to Switzerland during this time, in 1973), Speck’s laboratory and clinical work established immunosuppression with ATG as a treatment for patients with aplastic anaemia. Bone marrow transplantation using haploidentical donor bone marrow for SAA, after conditioning with ATG, proved effective. Today, it is known that ATG – rather than the transplanted cells – was responsible for this outcome. ATG has become a standard alternative to bone marrow transplantation for treating aplastic anemia. ^[15]

At Basel, the group around Speck belonged to the pioneers who treated patients with chronic myeloid leukaemia in the chronic phase with bone marrow transplantation. Together with investigators at the Royal Marsden Hospital [https://www.oncopedia.wiki/research-centres/royal-marsden], Speck and his Basel colleagues evaluated cyclosporine for the treatment and prevention of graft-versus-host disease. ^[16]

Bruno Speck and Jon van Rood created the European Group for Blood and Marrow Transplantation (EBMT). In 1975, Speck and van Rood organized a meeting of active investigators in BMT, which led to the establishment of EBMT. Speck was EBMT’s first president. In the early days, the “believers” of BMT reviewed cases and charts with each other and exchanged views on opportunities. In 1973, at the first gathering – still informal – the database comprised 13 patients. By 1990, 4025 transplantations were performed per year. ^[17]

The EBMT facilitated information sharing at an early stage, and then developed into a nonprofit organisation with the mission “To save the lives of patients with blood cancers and other life-threatening diseases by advancing the fields of blood and marrow transplantation and cell therapy worldwide through science, education and advocacy”.

Eliane Gluckman, a French haematologist, significantly influenced the field of bone marrow transplantation by realizing that cord blood is an unlimited source of hematopoietic stem cells for allogeneic HSCT. In 1989, Gluckman performed the first cord blood transplantation, transplanting a patient with Faconi’s anaemia with umbilical-cord blood from an HLA-identical sibling. ^[18] Initially, the use of umbilical cord blood was limited to children, as only a low cell dose could be infused. In the paediatric setting, cord blood transplants from both related and unrelated donors have been carried out with success. Several countries have set up banks for cord blood cells, which are HLA typed before being frozen. Studies suggest that Graft-versus-Host disease is reduced when umbilical cord blood cells are used for transplantation, instead of bone marrow. ^[19]

Refinement of transplantation: Graft-versus-host and graft-versus leukaemia.

European researchers contributed to refining bone marrow transplantation methods, notably to managing graft-versus-host disease.

One major complication of hematopoietic stem cell transplantation is an immunological-based complication, for which the term “secondary disease” was coined (to distinguish it from the primary sickness of radiation exposure), and which is now known as graft-versus-host disease (GvHD). From his clinical trials, Mathe termed the graft versus-host reaction, or secondary disease, "a stumbling block in the treatment of leukaemia by whole-body irradiation and transfusion of allogeneic hematopoietic cells. ^[20]

”Mathe and colleagues also described the graft-versus-leukaemia-effect, a preclinical observation that GvHD in mice resulted in the leukaemia cells being eradicated.

Van Bekkum and de Vries reviewed early attempts to control the graft-vs-tumour reaction, and early clinical work – almost all attempts failed. Van Bekkum and de Vries wrote: “It seems to be extremely difficult to induce that precise degree of graft versus host reactivity which will kill the leukemic cells but which is at the same time mild enough to allow survival of the host.” ^[9]

In the 1970s, Weiden and colleagues in Seattle demonstrated the correlation between GvHD and an antileukemia effect in humans (1979 for acute GvHD ^[21] and 1981 for chronic GvHD. ) ^[22]

The relapse rate of patients who had experienced acute GvHD was 2.5 times lower than the relapse rate of patients who had not experienced GvHD. However, the benefit for overall survival – stemming from the lower incidence of relapse – was offset by a higher mortality not associated with relapse in patients with GvHD.

GvHD was a double-edged sword, combining toxicity and cure. This sparked preclinical and clinical research to separate the harmful graft-versus-host disease from the beneficial graft-versus-leukaemia effect (GvL), and to develop strategies to reduce GvHD without increasing the rate of relapse.

One of the early pioneers in the field who had a strong influence on further research on GvHD and GvL was Milivoj Boranić (fig. 1.) from the Rudjer Boskovic Institute in Zagreb, Croatia. Boranić, as well as several colleagues from Zagreb, explored the temporal aspects of GvHD and GvL.

Boranić recalls how his interest in studying GvHD and GVL arose: “At the time, it was disputed by many scientists, claiming that it will not be possible to control the reaction of the transplant against leukaemia without mitigating the transplant’s power against leukaemia. My mentor [V. Stankovic] suggested to me an interesting approach would be to treat reaction in the host, so that the transplant loses its ability to harm the host but retains its effect against leukaemia. He suggested ‘it would be an interesting scientific approach for your thesis’ and it was the work for my PhD thesis in Zagreb, to study the effect of the graft against the host.”

Figure 1. Milivoj Boranić - Dubrovnik, September 2003.

Boranić recalls, however, that there were not enough mice with leukaemia in Zagreb to work with at the time. With an IAEA fellowship, Boranić was able to work in Rijswijk, Netherlands.

Boranić identified lymphohematopoietic tissues as the tissues first targeted in the GvH reaction. Boranić believed it was not necessary that the potentially lethal donor graft was present permanently and thought that the graft could be replaced by a less aggressive one after the leukaemia cells had perished, in a procedure which he called rescuing the host. Boranic later made attempts to induce an only transient GvH reactivity in dogs, and others in monkeys. ^[23]

This work by Boranić et al factually laid the foundation of the idea that cellular immunotherapy can cure cancer. The idea of “rescuing” the host through a less aggressive cellular therapy ^[24] was incredibly visionary and came to fruition years later in the form of donor lymphocyte infusions (DLI) by Kolb et al (see later) and ultimately the most modern approaches by genetically engineered CAR T cells. ^[25]

As stated, the work by Weiden et al. ^{[21] [22]} showing in transplanted patients a strong correlation between GvHD and the anti-leukemic effect of hematopoietic stem cell transplantation - which demonstrated the crucial effect of donor T cells in eliminating leukaemia cells - was the rationale for donor leukocyte infusion. Hans-Joachim Kolb (fig. 2.) and colleagues used donor T cells after transplantation to treat relapse in chronic myeloid leukaemia and other diseases. The donor leukocyte infusion resulted in impressive remission rates. ^[26]

Fig 2. Hans Joachim Kolb - Dubrovnik Meeting, September 2003.

Kolb achieved sustained remissions in patients with chronic myeloid leukaemia and, to a lesser extent, in patients with ALL by transfusing leukocytes from the original donor. Kolb had originally turned to hematopoietic stem cell transplantation out of interest in immune therapy. “I was looking for a thesis about immune therapy. Immune therapy is a big topic now, but back then, there was no immune therapy. But a professor worked on bone marrow transplantation – what is now referred to as stem cell transplantation. He said: ‘here, you have more immune therapy than with anything else’. And that’s true still today, I am convinced.”

Those animals who received lymphocytes from healthy donor, the host cells disappeared, and the animals turned into complete chimeras. This was not immediate but occurred over the course of months. This surprised us, and we tried if we could transfer immunity against infection, which was the case. It was a success, because we had a full chimerism with only donor cells, and because we could transfer immunity and induce new immunity effectively. This led us to do similarly in patients with a recurrence of AML or CML. We said – maybe this could also work in patients? We gave the patients lymphocytes from the donor. And over the course of weeks, the patients turned negative. So even with PCR we could no longer detect leukaemia cells. Initially people didn’t believe it, but then others repeated it and found similarly, which gave us some prestige. So, this was the first immune therapy without renewed chemotherapy or radiation therapy, which resulted in a long-term remission.” Another issue Kolb tackled was the recurrence of leukaemia after bone marrow transplantation. “When patients had a recurrence, they of course had many donor cells, including the normal haematopoiesis. In full recurrence, they reacted with bone marrow failure. For most patients we then, by giving them donor stem cells, could totally reverse the bone marrow failure. This was a logical consequence, which made us very happy.”

Finally, Kolb was able to realize his initial wish of developing an immune therapy. ^[27]

“With donor lymphocytes, one is able to do immune therapy. So we don’t have to eradicate every last leukaemia cell, but with the functional donor lymphocytes, we can do immune therapy. These cells are accepted by the host, because he already has the donor immune system, so there will be no rejection of donor lymphocytes. This is a huge success.”

Despite significant clinical advancements aimed at reducing Graft-versus-Host Disease (GvHD) and optimizing the Graft-versus-Leukemia (GVL) effect, as initiated by Weiden's groundbreaking paper, achieving a definitive separation between GvHD and the GVL effect remains the elusive goal of allogeneic stem cell transplantation. However, the GVL effect stemming from blood and marrow transplantation has emerged as the most successful form of adoptive cellular immunotherapy. BMT has evolved into an effective platform for delivery of cellular immune therapies and not simply for delivery of high-dose chemotherapy.

HSCT in Zagreb and ESO Meetings

At the end of 70-ies of the 20th century HSCT has become a routine treatment programme in Western well-developed European countries. In Eastern Europe transplantation was still a “mission impossible”. Soon, two moments strongly facilitated the activities for launching the transplant programme in Zagreb, Croatia. First, it was previously mentioned, scientific work of the Zagreb experimental bone marrow transplant team at the Ruđer Bošković Institute. The physicians were well informed about the new potent therapy for malignant disorder. They also recognized enormous and quick implementation of HSCT in developed countries. Secondly the resources were partly covered by the Krško Nuclear Plant, which was under construction at the time, since bone marrow transplantation was planned as part of the tertiary medical care for the plant. HSCT was not a priority in Croatian medicine. Many physicians doubted the program, arguing that therapy for a few patients was not as important as, for example, vaccination for all. At that time, it was not able to recognize the importance of transplantation especially the influence on the development of clinical medicine. Therapy for only a few patients was not a strong argument to convince the majority that transplant programme is a priority. ^[28]

Following the training of Zagreb's physician in Paris (E. Gluckman), Seattle (D. Buckner) and Basel (A Gratwohl) HSCT was started in 1983. In a few years HSCT in Zagreb has become a routine therapy. The results were very encouraging almost identical to those in well developed countries. Zagreb transplant team recognized another important aspect for optimal development of transplant programme and haematology.

Figure 3. 1st Meeting "New trends in the Treatment of Acute Leukaemia" in Dubrovnik, September 1987.

There was a need for closer contact with the well-known scientists and physicians from abroad. The way how to realise it could be scientific meetings with transplantation as a specific topic. The international meetings in Europe and USA were too broad covering many scientific topics. The intention was to organize such meeting in Croatia, every 2-3 years covering transplantation and acute leukaemia. European School of Oncology [https://www.oncopedia.wiki/cancer-organisations/eso] (ESO) from Milan and its Secretary General Alberto Costa recognized the importance of such meetings and together with Zagreb School of Medicine supported organizing the scientific conference named “New trends in the treatment of acute leukaemia”. These meetings started in 1987 in Dubrovnik (fig. 3.).

Figure 4. B. Labar and S.Z. Pavletic, at the American Society of Hematology annual meeting in Philadelphia, 2002

Boris Labar and Steven Pavletic (fig. 4.) were the local organizers. The success of the Meeting was beyond all expectations, so the second one was organized after two years (fig. 5 and 6.), with twice more participants (about 300) compared to first one. Besides the transfer of new relevant information, the meeting facilitated the contact between leading world haematologist and participants from Eastern European countries.

Figure 5. 2nd Dubrovnik Meeting September, 1989.

Figure 6. The Poster of Dubrovnik Meeting

Timetable of the meetings, together with development of Zagreb transplant programme is given in fig. 7.

Figure 7. Timetable of the New Trends Meetings and Zagreb Transplant Programme. The figure illustrates the impact of ten consecutive conferences under auspices of ESO since 1987 “New Trends in the Treatment of Acute Leukemia” and the corresponding introduction of new transplant technologies by the Zagreb team. These conferences spanning almost over two decades remained the main venue for the dissemination and advancing tretments of haematological malignancies in Eastern Europe.

Zagreb program and scientific meetings New Trends, as depicted in figure, have been growing very similarly and influenced each other. These meetings continued in early 1990s despite of the war of independence situation in Croatia. Meeting was displaced in the western part of Croatia on safe distance from war zone. In the recent years the meeting changed the name into “Leukaemia and Lymphoma”. Lymphoproliferative disorders were also included as a topic.

Currently Zagreb transplant team have been performing all the treatment approaches related to HSCT. ^{[29] [30]}

The Zagreb chronic GvHD program to address transplant complications in increasingly growing number of long-term survivors after successful transplantation formed in 2013 became one of leading such canters in the World. ^[31]

Back in 1980-ies when the transplant programme has started, the support of medical professionals and societies were crucial, ^[28] At that time the transplantation was rare in Eastern Europe, even in the Western Europe and USA.

The best way to illustrate those days and the problems Zagreb team had faced at the beginnings might be the opinion of one of the leading persons in the field of transplantation Dean Buckner from Seattle, USA. During his visit of Zagreb Centre in 1980s someone asked him: “What is your opinion concerning Zagreb transplantation?” - he simply answered: “It's a miracle”.

Not only Dean Buckner but also many others supported Zagreb programme. ESO made a major contribution that programme was recognized worldwide. By organizing the meeting in Dubrovnik ESO also facilitated international cooperation with leading world transplant centres and sped up the starting of the other new transplant centres in Eastern Europe.

Conclusion

Scientific collaboration through scientific meetings initiated by ESO and Zagreb team had a great impact on development of HSCT in Croatia and Eastern Europe. It also contributed to the international recognition of the programs. Today after 40 years and more there is no doubt that HSCT had a significant influence on the progress of clinical medicine across Europe. As depicted in this article, European scientists, clinical researchers and above all our patients and families, made major contributions to the evolution of the bone marrow transplantation as one of most important advances in the modern medicine.