Allogeneic Bone Marrow Transplantation for Aplastic Anemia

Abstract

After more than 60 years of intense research in allogeneic hematopoietic cell transplantation (HCT), this therapy has progressed from one that was fraught with seemingly insurmountable complications to a standard treatment of patients with aplastic anemia. During the 1970s and 1980s, HCT donors were almost exclusively HLA-identical siblings. Subsequent advances in the understanding of the complexity of the HLA region along with the development of molecular HLA typing and the establishment of unrelated volunteer donor registries have resulted in an ever-increasing use of such donors. Most recent breakthroughs have enabled HLA-haploidentical HCT and, thereby, finding donors for nearly every patient. The outstanding outcomes reported with any of the donor options have made allogeneic HCT the preferred treatment over immunosuppressive therapy.

1. Early Challenges

The history of allogeneic marrow transplantation for otherwise lethal bone marrow-based diseases including marrow failure syndromes, such as aplastic anemia, began after the second World War. The issue of marrow failure came into focus after the atomic bomb explosions at Nagasaki and Hiroshima in 1945. The late deaths from marrow failure in individuals distant from the epicenters of the bomb explosions alarmed the military. They became concerned about soldiers exposed on the battlefield to nuclear weapons dying from marrow aplasia. Therefore, much funding was spent on investigations on how to protect soldiers from irradiation-induced marrow failure and death.

It was not until 1949 that a scientific paper reported the landmark finding that mice could be protected from lethal total body irradiation injury to the marrow by exteriorizing and then shielding their spleens with lead [1]. Two years later, in 1951, it was shown that mice and guinea pigs could survive marrow failure after total body irradiation by subsequent infusion of syngeneic marrow cells [2]. Of note, these early authors posited that humoral factors in the spleen and the marrow induced the rapid post-irradiation marrow recovery.

However, in the ensuing years, an increasing number of investigators began questioning this “humoral hypothesis”. Finally, in 1955/1956, several laboratories in the United Kingdom, the Netherlands, and the United States, using blood genetic markers, provided unequivocal proof in mice and rats that the life-saving effects of marrow or spleen cell infusions were due to a cellular mechanism [3–5]. These findings definitively refuted the humoral hypothesis and firmly established a cellular origin of the hematopoietic recovery after irradiation.

The findings, made in rodents, provided a starting point for allogeneic marrow transplantation in humans, not only for the treatment of individuals exposed to accidental irradiation but, more importantly, to treat patients with fatal malignant and non-malignant blood disorders, such as leukemia or aplastic anemia. Unexpectedly but, in retrospect, not surprisingly, the translation from inbred rodents to random-bred humans turned out be a complete failure. A paper from 1970 summarized the results from 200 reported patients transplanted for hematopoietic malignancies or marrow failure, none of whom survived [6]. These included 73 patients transplanted for aplastic anemia, 66 of whom died with graft failure, 5 with graft-versus-host disease (GVHD), and 2 from other causes. Likely, the unsuccessful published cases represented only the tip of the proverbial ‘iceberg’, and many more patients died from failed marrow transplantations.

These early human marrow grafts were attempted before a full understanding of conditioning regimens, the risk of graft rejection due to patient sensitization to donor antigens via blood transfusions, and GVHD prevention was achieved, and before the discovery of the importance of in vitro histocompatibility matching for graft outcome.

As a result of the uniform failure of early human transplantation, most investigators left the field. As van Bekkum and de Vries stated in their 1967 book, Radiation Chimeras, “Investigators abandoned the idea that marrow transplantation could ever become a valuable asset of clinical medicine” [5]. The problems encountered seemed insurmountable.

2. Back to the Preclinical Domain

However, undeterred by the early, disastrous, clinical marrow transplantation outcomes, a few small laboratories in Europe and the United States persisted in efforts of understanding and overcoming the perceived “insurmountable” obstacles encountered in early transplantations. Much of the ensuing work was done in large animals including monkeys and dogs. A paper from 1968 reported the new and important observation that canine littermates matched for the major histocompatibility complex (MHC) antigens by in vitro tissue typing had far better outcomes than MHC-mismatched littermates [7]. Significantly better outcomes were also seen among unrelated, MHC-matched canine marrow graft recipients compared to their MHC-mismatched counterparts [8]. MHC typing in those days was primitive and consisted of serologic measurements using multi-specific antibodies, harvested from the blood of multiparous individuals, in tricky trypan blue exclusion or leuko-agglutination assays [9–12]. Serological results were combined with those obtained from testing donor and recipient lymphocytes for reactivity in an equally fickle mixed leukocyte culture assay as a way of determining MHC identity [13].

A second, completely unexpected, observation made in the canine experiment was that fatal GVHD, either acute, subacute, or chronic, developed in MHC-matched littermate recipients, even though it occurred significantly later than in their MHC-mismatched counterparts [7]. This finding was not at all anticipated from previous work in mice or rats. It emphasized the need for developing methods to prevent and control GVHD, even in well-matched donor/recipient combinations.

Consequently, tedious investigations of numerous, then available immunosuppressive agents were conducted in the canine model. These eventually led to identifying an antimetabolite, methotrexate (MTX), as the best drug for GVHD prevention [8, 14]. Balancing the drug’s toxicities against its efficacy, a regimen of intermittent MTX dosing was developed. For achieving greatest efficacy with least toxicity, the drug was administered 1, 3, 6, and 11 days after transplantation, and then weekly for at least 3 months.

Other efforts focused on identifying effective and tolerable conditioning regimens. In competition and collaboration with a colleague and friend at Johns Hopkins Medical School, the late George Santos, we investigated cyclophosphamide (Cy) as a conditioning agent. We found it effective for enabling engraftment of DLA-identical littermate marrow, resulting in stable long-term mixed donor-host chimerism. This, we thought, would make Cy an ideal conditioning regimen for patients with aplastic anemia whose marrow was empty while their immune system was intact. The final Cy conditioning regimen, suitable for translation into the clinic, was worked out in rhesus monkeys. Cy, given at 50 mg/kg body weight per day on 4 consecutive days, enabled successful allogeneic marrow grafts in randomly selected, unrelated primate recipients [15]. Of note, at this Cy dosing, monkeys not given marrow grafts experienced profound pancytopenia, but promptly and completely recovered their blood counts within three to four weeks, indicating that the drug did not damage hematopoietic stem cells while exhibiting excellent immunosuppressive properties.

Other studies in dogs addressed transfusion-induced sensitization to minor histocompatibility antigens, which placed patients at risk of marrow graft rejection. We found that giving dogs three preceding, 50-ml whole blood transfusions from their DLA-identical littermates resulted in 100% rejection of subsequent marrow grafts from the transfusion donors [16]. With nine transfusions from unrelated donors, the rejection rate was 35%, indicating that a third of these unrelated transfusion donors shared minor histocompatibility antigens with given marrow donors but not with their respective DLA-identical littermate recipients [17]. We further found that removing leukocytes from platelet and red blood cell transfusion products reduced the rejection rate to 35% [18]. Most importantly, we made the serendipitous observation that treating transfusion products with 1500 cGy in vitro irradiation reduced the rejection risk to 10% [19]. After some lag time, these straightforward and simple methods have now become standard blood bank procedures.

Equally importantly, a more immunosuppressive and effective conditioning regimen was developed in the canine model that gave rise to the clinically used anti-thymocyte globulin (ATG)/Cy regimen [20].

3. Return to the Clinic: 1970 to 1985

In 1970, the first successful marrow transplant from an HLA-identical sibling was done in an aplastic anemia patient. The Cy conditioning regimen was identical to that published in rhesus monkeys in the same year [15]. Post-grafting immunosuppression included intermittent MTX, developed in the canine model and published in 1970 [14]. At that time, HLA typing methods and the understanding of the MHC were both primitive and incomplete. Only one locus was recognized, which was called HL-A. The early patients were matched serologically for this locus with their siblings. Confirmation of the serological match was obtained by mixed leukocyte culture non-reactivity. A paper in 1972 summarized the outcomes among the first four cases [21]. One of the four patients died of GVHD, one died after graft rejection, and two became long-term survivors with sustained donor cell engraftment. The longest surviving aplastic anemia patient, transplanted in 1971, is well with normal hematopoiesis of donor origin.

The subsequent, early Seattle experience showed that marrow graft rejection was a major obstacle to success [22, 23]. Thirty-three percent of multiply transfused aplastic anemia patients rejected their grafts. This contrasted with an ~8% rejection rate among untransfused patients. This observation validated the concerns raised by the preceding canine studies on the effect of preceding blood transfusions. Other centers reported equally high or even higher graft rejection rates [24]. Graft rejection in these early years was associated with a very high mortality rate.

The second major cause of mortality was complications of acute and chronic GVHD with a rate of 15%. As a result of the cumulative mortality from both rejection and GVHD, 12-year survival in the first group of 81 transfused patients was 46% compared to 80% among 39 untransfused patients (Figure 1). Very similar results, with shorter observation times were reported by the Marrow Transplant Registry [24].

Figure 1.

Kaplan-Meier product limit estimates of percent surviving for Seattle patients treated by Cy and marrow grafts from HLA-identical siblings [25]. Shown are data in 43 untransfused patients given marrow only, 65 transfused patients given marrow and buffy-coat ceil infusions, and 93 transfused patients given marrow only. Day 0 is the day of marrow grafting. Tick marks indicate surviving patients. Survival is as of June 10. 1983. Figure reused with permission from Storb R, et al. Marrow transplantation for aplastic anemia. Seminars in Hematology. 1984;21(1):27–35.

In 1977, the Seattle team published an analysis of factors associated with graft rejection in multiply transfused patients, who were conditioned with Cy [23]. Among the many factors studied, the strongest association was with a unidirectional positive response in mixed leukocyte culture with patient cells responding to donor cells and not the other way around. This finding was interpreted as an expression of sensitization of the patient against non-HLA antigens of the donor through transfusions. Another risk factor for graft rejection was a low transplanted marrow cell dose. To overcome that limitation, based on preceding studies in rodents, dogs, rhesus monkeys, and baboons showing the presence of hematopoietic cells in the circulating blood, we attempted increasing the number of transplanted stem cells in transfused patients by adding several days of donor buffy coat cell infusions to the marrow inoculum. Indeed, with this strategy, the graft rejection rate declined to 8%, and patient survival increased to over 70% (Figure 1; [26]). However, this advance was achieved at the expense of a dramatically increased rate of chronic GVHD.

Other transplant centers pursued different approaches at decreasing the risk of graft rejection with varying degrees of success [27–33] (Table 1). Ramsay et. from the Minnesota group combined Cy with 750 cGy total lymphoid irradiation (TLI). This increased survival to 72%. Gale et al. from UCLA and Elfenbein et al. from Johns Hopkins added total body irradiation (TBI) and saw survivals of 62% and 39%, respectively [28, 29]. Parkman et al. from Harvard added procarbazine to Cy as previously described by the Seattle team [20] and saw 64% survival [31]. Similar success was reported by the European Bone Marrow Transplant Registry. The various European groups reporting to the Registry used Cy combined with either TLI, total body irradiation (TBI), or thoraco-abdominal irradiation (TAI) [34]. An example was the report by Devergie et al which reported 78% survival [27]. Overall, efforts to reduce the graft rejection risk by adding some form of radiation resulted in improvement of survival to 63%. Similar data were reported by the International Bone Marrow Transplant Registry (IBMTR).

Table 1.

Early results with marrow grafts from HLA-identical siblings for multiply-transfused aplastic anemia patients

Investigators [reference]	Number of patients	Age in years (median)	Conditioning regimen*	Rejection† (%)	Dead with GVHD† (%)	Alive (%)	Follow-up
Parkman et al. [31]	14	2–31 (18)	PA + CY	15	18	64	4.5 mo to 5 yr
Ramsay et al. [32, 33]	40	1–40 (13)	CY + 7.5 Gy TLI	10	18	72	1.5 mo to 5 yr
Elfenbein et al. [28]	75	5–49 (20?)	Cy ± PA ± TBI	43	?	39	3 mo to 8 yr
Gale et al.‡ [29]	59	3–43 (17)	Cy + 3 Gy TBI	5	31	62	6 mo to 5.5 yr
Devergie and Gluckman‡ [27]	28	6–33 (15)	Cy + 6 Gy TAI	5	14	78	3 mo to 2.25 yr
Hows et al.‡ [30]	35	7–39 (20)	Cy	14	15	77	2 mo to 2.5 yr
Seattle [26, 25]	65	3–53 (21)	Cy	13	15	71	6 mo to 7 yr

^*Abbreviations: Cy = cyclophosphamide; PA = procarbazine and antithymocyte globulin; TAI = thoracoabdominal irradiation; TBI = total body irradiation; TLI = total lymphoid irradiation

^†Percentage refers to patients at risk.

^‡Data were updated through personal communications from Drs. Gluckman, Hows, and Gale.

Table reused with permission from Storb R, et al. Marrow transplantation for aplastic anemia. Seminars in Hematology. 1984;21(1):27–35. © Elsevier.

Age 0–20: 86 (BMT), 84 (IST)

Age 21–40: 76 (BMT), 65 (IST)

Age >40: 56 (BMT) 58 (IST)

One problem with the use of irradiation-based regimens has been secondary malignancies after transplantation. A 20-year rate of secondary malignancies of 14% was reported among patients given Cy combined with various forms of radiation [35], while patients receiving Cy only had a cumulative 25-year incidence of only 7% [36]. In the latter setting, tumors were largely skin and oropharyngeal tumors that occurred with a delay of at least 14 years; tumor development was statistically significantly associated with chronic GVHD but not with conditioning with Cy.

4. Moving On: 1986 through 1990s

As already indicated, studies in the canine model established a conditioning regimen that promised to overcome transfusion-induced sensitization to donor antigens and reduce the risk of graft rejection far better than Cy alone. This regimen was translated into the clinic in the form of a highly immunosuppressive combination of Cy alternating with ATG. The Cy/ATG regimen was first evaluated in conditioning patients for second, salvage transplantation [37]. After finding it highly effective, we introduced Cy/ATG for upfront transplantation in 1988 [38]. At that time, added buffy coat cell infusions were discontinued. With the Cy/ATG regimen, graft rejections among aplastic anemia patients given HLA-identical marrow grafts have become the exception, and overall survival has improved markedly. Also, in part because buffy coat cell infusions were abandoned, chronic GVHD markedly declined to a cumulative incidence of 16%. A case-matched comparison published in 1994 showed 92% 8-year survival for Cy/ATG-conditioned patients compared to 72% survival for Cy-conditioned patients [38]. Over the past 12 years, survival at our center has been 100%, and the Cy/ATG regimen has become the standard of care for HLA-matched related marrow grafts.

However, at around the time of the introduction of the Cy/ATG regimen, blood banks began providing leukocyte-poor transfusion products. Also, not long thereafter, transfusion products for marrow transplantation candidates were routinely being irradiated in vitro. This raised the question whether the reduced rejection rates seen in more recently transplanted patients were due to the Cy/ATG regimen or the almost simultaneously introduced changes in blood transfusion practices, which, experimentally, had been shown to reduce the risk of sensitization to minor histocompatibility antigens via transfusions and, along with it, the risk of graft rejection.

A multi-center, cooperative study coordinated by the IBMTR tried to address this question. The trial randomized patients between the Cy/ATG regimen and Cy alone [39]. Unfortunately, the study had difficulties accruing patients. After a 7-year period of sluggish enrollment, it was concluded prematurely. Thus, the trial’s answer to the question posed was not definitive. Graft rejection rates in the two groups of patients were comparable. Five-year survival was 80% in the Cy/ATG group compared to 74% in the Cy group, a difference that was not statistically significant.

Through the 1970s, intermittent MTX was the predominant immunosuppressive drug used for GVHD prevention. Canine studies, published in the early 1980s showed that a combination of a short course of MTX (days 1, 3, 6, and 11) and an extended course of cyclosporine (CSP) had synergistic immunosuppressive effects, and GVHD prevention was highly significantly improved [40]. Two subsequent prospective, randomized clinical trials, one in aplastic anemia patients undergoing HLA-matched related transplants, and the other among patients with acute or chronic myeloid leukemias, confirmed that the combination of MTX and CSP was significantly better than either drug alone [41, 42]. Results of these trials were published in 1986. Since then, MTX combined with CSP (and later also with tacrolimus (TAC)) has remained a mainstay of post-grafting immunosuppression for prevention of acute GVHD.

Following the introduction of the MTX/calcineurin inhibitor combination, overall survivals among aplastic anemia patients given HLA-matched related grafts have improved. Publications from the 1990s by the CIBMTR, the EBMT, and individual marrow transplant centers reported survivals ranging from 64% to 88% (Table 2). An interesting and effective, novel approach at transplantation has been published by physicians in the United Kingdom [43]. They combined fludarabine (Flu)/Cy conditioning with alemtuzumab (CAMPATH), followed by post-grafting immunosuppression with CSP only and saw overall survivals of 88%.

Table 2.

Select recent reports of HLA-matched sibling and unrelated donor hematopoietic cell transplantation for severe aplastic anemia.

Reference	Year of report	Year of transplant	Number of patients	Age range in years (median)	Conditioning program*	Hematopoietic source	Prevention of GVHD	Graft rejection/ failure (%)	GVHD (%)		Survival (%)	Range of follow-up in years (median)
									Acute	Chronic
HLA-identical sibling donor transplantation
Burroughs et al. [44]	2012	1971–1984 1981–1988 1989–2010	98 19 31	1.8–19 (12.8)	CY CY CY+ATG	BM	MTX CSP+MTX CSP+MTX	22 32 7	21 11 39	21 21 10	66 95 100	11–37.2 (31.1) 15.2–27.1 (23) 0.3–21.5 (6.1)
Bacigalupo et al. [45]	2012	1999–2009	1886	1–68 (18)	CY+ATG (41%), and various	BM n=1163	CSP+MTX (41%), or other	9	11	11	Age ≤ 20: 90 Age > 20: 74	(2.1)
				1–69 (24)		PBSC n=723		10	17	22	Age ≤ 20: 76 Age > 20: 64	(2.0)
Maury et al. [46]	2009	1998–2007	30	31–66 (46)	Flu+CY+ATG	BM n=20 PBSC n=10	CSP+MTX or other	3	10	13	77	1.1–6.8 (4.1)
Bacigalupo et al. [47]	2016	1999–2014	BMT n=1732 IST n=802	0–20 21–40 >40	BMT-various	Compare BMT vs. IST	Various for BMT	N/A	N/A	N/A	BMT: Age 0–20: 86 Age 21–40: 76 Age >40: 56	0.2–10
					IST-various						IST: Age 0–20: 84 Age 21–40: 65 Age >40: 58
Bacigalupo et al. [48]	2015	2005–2009	940	age >20 yr.: 50%	various	BM =566 PBSC =374	various	9	13	14	Low risk 93 Int. risk 78 High risk 67	0.1–9.1(3.1)
Gallo et al. [49]	2016	2006–2015	21	3–52 (15)	CY+ATG	BM <2.5×108 TNC/kg	CSP+MTX	0	47	16 (24)	100	1–8 (4.0)
Unrelated donor transplantation
Samarasinghe et al. [50]	2012	2000–2010	44	3–19 (8)	Flu+CY+Alemtuzumab various doses	BM=26 PBSC=18	CSP or CSP+ MMF or MTX	0	38	12	95	1–6.3 (2.9)
Bacigalupo et al. [48]	2015	2005–2009	508	age >20 yr.: 53%	various	BM=264 PBSC=244	various	9	25	26	Low risk 83 Int. risk 77 High risk 64	0.1–9.1(3.1)
Dufour et al. [51]	2015	2005–2014	29 upfront MUD HCT (no prior IST)	1.7–19.1 (8.4)	Flu+CY+Alem (+2–3 Gy TBI for 1 Ag-MM)	BM=21 PBSC=8	CSP or CSP/MMF	4	10	19	96 OS 92 2-yr EFS	0.2–8.5 (1.7)
Anderlini et al. [52]	2015	2006–2013	79	0.5–66 (24)	ATG+2 Gy TBI+ CY-100 (n=41) vs. CY-50 (n=38)	BM	CSP/MTX	CY 100: 15	27	32	81 @ 1 yr	1.0–4.2 (2.0)
								CY 50: 12	24	23	97 @ 1 yr	0.3–2.2 (1.4)
Marsh et al. [53]	2014	1999–2009	55 URD	1–67 (18)	Flu+CY+Alem	BM =36 PBSC=19	Tacrolimus/MMF	9	38	13	88 97 BM 70 PBSC	0.5–10

HLA= human leukocyte antigen; GVHD= graft-versus-host disease; CY= cyclophosphamide; ATG= anti thymocyte globulin; BM= bone marrow; MTX= methotrexate; CSP= cyclosporine; PBSC=peripheral blood stem cells; Flu= fludarabine; IST= immunosuppressive therapy (non-transplant); TNC= total nucleated cells; kg= kilogram; MMF= mycophenolate mofetil; yr.= year; Int.= intermediate; MUD= matched unrelated donor; HCT= hematopoietic cell transplantation; Alem= alemtuzumab; Gy= Gray; TBI= total body irradiation; 1 Ag-MM= One HLA-antigen mismatch; OS= overall survival; EFS= event-free survival; CY-100= cyclophosphamide 100 mg/kg; CY-50= cyclophosphamide 50 mg/kg; @ 1 yr= survival at 1 year after transplant; URD= unrelated donor.

Reused by permission from author (R. Storb) from Georges GE et al. Severe aplastic anemia: allogeneic bone marrow transplantation as first-line treatment. Blood Adv. 2018;2(15):2020–8. doi:10.1182/bloodadvances.2018021162. © 2018 Elsevier

In line with the changes in conditioning regimen and GVHD prevention, outcomes of HLA-identical sibling transplantation for pediatric patients gradually improved over time as illustrated in a retrospective analysis from Seattle which showed 66% overall survival in an early cohort of patients transplanted between 1971 and 1984 compared to 95% survival in a cohort transplanted from 1984 through 1988, and 100% survival among subsequent patients [44].

Additional major contributors to improved survivals seen at all transplant centers have been advances in supportive care, especially in infectious diseases. For example, prophylactic acyclovir has prevented the not infrequently fatal reactivation of the varicella zoster virus. Monitoring and preemptive treatment of cytomegalovirus reactivation with ganciclovir or foscarnet and the recent introduction of prophylactic letermovir, have decreased the risk of dying from cytomegalovirus infections. Other important advances have been made in the treatment and prophylaxis of fungal and bacterial diseases. The introduction of ursodiol for protecting the liver against chemo-radiation damage had the serendipitous side effect of reducing the risk of liver GVHD

6. The 21^st Century

Is there an upper age limit for patients with aplastic anemia undergoing allogeneic marrow transplantation? This controversial question goes back to 1970 when the two current, competing treatment strategies for severe aplastic anemia, allogeneic marrow transplantation and immunosuppressive therapy (IST) with ATG were introduced [21, 54]. ATG-based IST was later augmented by CSP and, most recently, by eltrombopaq. Major problems with IST have included failure to respond, recurrence of aplastic anemia, and development of late myelodysplastic syndrome or acute myeloid leukemia [55]. An informal convention has been to treat younger patients by upfront marrow transplantation and older patients with upfront IST. The threshold age was around 40 years. Indeed, in the early years of transplantation for aplastic anemia, most patients were either children, adolescents, or young adults, and reported median ages ranged from 18–25 years. Older patients were transplanted only after IST failure; not surprisingly, such patients were often infected or platelet transfusion refractory when they underwent transplantation, setting the stage for poorer transplantation outcomes. These poorer outcomes, in turn, perpetuated the recommendation to use IST first in older patients.

A publication from 2010 addressed the issue of age and summarized the early Seattle transplantation experience among patients aged 40–68 years [56]. Ten-year survival among these patients was 65%. In part, the somewhat poorer long-term survival could be explained by the fact that close to 50% of the patient cohort had first been treated with and then failed IST. Undergoing marrow transplantation with a history of infections and long-term antibiotic use has been an adverse risk factor for transplant outcome. A retrospective analysis of CIBMTR and EBMT data, including close to 500 patients above age 50 years, confirmed the Seattle results and showed 3-year survival in the range of 60% [57].

However, more recently, the pendulum swung in the direction of marrow transplantation as the most effective upfront therapy, not only in younger patients but also in older patients.

In this respect, a retrospective analysis of pediatric EBMT and United Kingdom data was important and helped setting the stage for the eventual change [51]. The analysis showed 90% 7-year survival with unrelated marrow transplantation after conditioning with Flu/Cy/alemtuzumab compared to only 25% with IST. Equally important, the investigators showed 90% 7-year survival with upfront transplantation compared to only 70% with transplantation after failure of IST. These data illustrated the adverse effect of preceding IST on transplantation outcome and supported the concept of upfront transplantation, at least in younger patients.

Firm support for upfront transplantation in older patients was provided by a retrospective analysis from King’s College Hospital in London [58]. The investigators had used the Flu/Cy/alemtuzumab regimen to condition 27 patients, aged 51–71 years, and 38 patients, aged 17–49 years, for peripheral blood stem cell transplantation from either HLA-identical siblings or HLA-matched or one HLA antigen mismatched unrelated donors. GVHD-free and relapse-free survival at 5 years was 94% for the younger cohort and 84% for the older cohort, a difference which was not statistically significant (Figure 2). A retrospective review from EBMT also showed advantages of marrow transplantation over IST.

Figure 2.

Overall survival of patients according to age and cumulative incidence of GVHD. (A) OS for age older than 50 vs younger than 50 years. (B) Cumulative incidence of chronic GVHD for age 50 years or older vs younger than 50 years. Modified and reused with permission from Sheth VS, et al., Similar outcomes of alemtuzumab-based hematopoietic cell transplantation for SAA patients older or younger than 50 years. Blood Adv. 2019;3(20):3070–9. doi:10.1182/bloodadvances.2019000480. Copyright © 2019 by The American Society of Hematology.

Given the risks of failure to respond, recurrence of aplastic anemia, and late complications with IST [55, 59], it is therefore reasonable to recommend upfront transplantation in all patients with aplastic anemia, rather than transplantation after failure of IST. Whenever a patient is diagnosed with aplastic anemia, HLA typing of the patient should be done, and an urgent donor search be initiated.

An EBMT analysis published in Biology of Blood and Marrow Transplantation (BBMT) in 2012 showed survivals in patients less than 20 years of age of 90% compared to 74% in patients older than 20 years [45]. A subsequent retrospective analysis from EBMT published in 2015 showed survivals in both older and younger patients ranging from 67% to 93%, depending on the risk score [48].

Most marrow transplantations in the 20^th century were from HLA-identical sibling donors. An early report by Gordon-Smith et al. already showed the feasibility of using histocompatible unrelated volunteers as marrow donors for aplastic anemia patients [60]. With dramatically increased understanding of the complexity of the HLA region and associated advances in molecular HLA typing, as well as the development of national and international unrelated marrow donor registries, unrelated HLA-matched transplantations have increasingly been used. However, while unrelated transplant outcomes have improved, there are still differences with HLA-identical sibling transplantations. For example, an EBMT analysis showed that the incidence of acute and chronic GVHD was greater for unrelated recipients than for HLA-identical sibling recipients, 25% and 26% vs. 13% and 14%, respectively [48]. Accordingly, the overall survival after unrelated grafts was worse than after HLA-identical sibling grafts; however, the difference was not statistically significant.

Improvements in unrelated transplantation have, in part resulted from methods to reduce conditioning regimen toxicity. This has included combining a single dose of Cy with Flu, ATG, and low-dose TBI. A multi-center U.S. trial determined that the optimum conditioning regimen included horse or rabbit ATG, a single dose of Cy, 50 mg/kg, four days of Flu at 30 mg/m²/day, and 2 Gy TBI on day -1 [52]. With a median follow-up of 17 months, the 1-year survival rate was 97.4%. As already described, an alternative regimen with equally outstanding results has been to combine Cy and Flu with alemtuzumab [53].

While marrow was the graft source in all early transplantations, hematopoietic cells collected from the peripheral blood have begun displacing marrow for allogeneic transplantation, especially among patients with hematologic malignancies. However, the Seattle group has continued using marrow for patients with aplastic anemia, given a low incidence of chronic GVHD associated with that graft source. In support of this preference, trials using granulocyte colony-stimulating factor (G-CSF) mobilized peripheral blood mononuclear cells in patients with aplastic anemia have consistently shown higher rates and greater severity of both acute and chronic GVHD and, consequently, worse survival [45, 61, 62]. The exception has been patients conditioned with an alemtuzumab-based regimen where low rates of acute and chronic GVHD have been reported [53].

While easy access to the HLA data from > 40 million unrelated volunteer donors in the various registries (The Worldbook) has enabled the ever-increasing use of such donors, donor searches have encountered one serious limitation: until recently, most donors in the Worldbook have been of Caucasian origin. As a result, greatest success in finding a suitable donor has been with Caucasian recipients, while finding donors for recipients from minority populations has been difficult.

A breakthrough in overcoming the ‘donor bottleneck’ occurred in the 2000s when a minimal-intensity conditioning regimen was paired with novel GVHD prevention, thereby enabling successful HLA-haploidentical related marrow transplantation. This breakthrough meant that nearly every patient with aplastic anemia had a potential marrow donor. How did the regimen evolve? Investigators at Johns Hopkins Medical School augmented pre-transplant immunosuppression of a minimal-intensity conditioning regimen of Flu and 2 Gy TBI, originally developed by Seattle investigators [63], with two small doses of pre-transplant Cy, 14.5 mg/kg/day on days −6 and −2 and ATG from days -9 to -7 [64]. For GVHD prevention, they used post-transplant immunosuppression with two high doses Cy, 50 mg/kg/day, on days +3 and +4. The latter use of Cy was first reported by Japanese investigators who demonstrated in mice that post-transplant Cy got rid of rapidly proliferating, transplanted donor lymphocytes, activated against host antigens (reviewed in [65]). The removal of the host-responsive donor lymphocytes by CY reduced both the incidence and the severity of GVHD. For the human protocol, the Johns Hopkins investigators added a 35-day course of mycophenolate mofetil and an extended course of a calcineurin inhibitor after the day +4 CY; the synergistic combination of mycophenolate mofetil (MMF) and CSP was introduced earlier by the Seattle group since it both enhanced engraftment and controlled GVHD in MHC-matched grafts [63].

The novel approach at HLA-haploidentical transplantation has been surprisingly successful and is now widely used for patients with hematologic malignancies and non-malignant blood disorders. It has broadened the application of hematopoietic cell transplantation to include patients from previously underserved minority populations. HLA-haploidentical children, parents, siblings, or other relatives can serve as donors.

Early results with HLA-haploidentical related marrow transplantation in aplastic anemia patients have been impressive. A publication from 2017 reported outcomes among 16 patients aged 21–69 years [66]. At the time of the report, all 16 patients were alive with hematopoietic engraftment. More recent updates of the early results [64, 66] including results from a multicenter trial using the Johns Hopkins approach [67], confirmed the early results, with 1-year survival in the multi-center study of 81% among 32 patients.

A review of EBMT data also showed excellent outcomes of HLA-haploidentical transplantation among a small number of aplastic anemia patients given the regimen developed by Johns Hopkins while patients given other regimens fared far worse [68].

A different approach at HLA-haploidentical related transplantation for patients with aplastic anemia has been reported by Chinese investigators [69]. They conditioned their patients for transplantation with busulfan, 3.2 mg/kg/day on days −7 and −6; Cy, 50 mg/kg/day on days −5 to −2; rabbit ATG, 2.5 mg/kg/day on days −5 to −2. GVHD prevention consisted of a short course of MTX (days 1, 3, 6, and 11), MMF through day 60, and CSP through one year. They enrolled 392 patients. Among the 381 patients who lived more than 28 days, 379 engrafted. Two-year survival after grafts from fathers, mothers, siblings, and children were 86.6%, 87.1%, 84.3%, and 92.2%, respectively. Acute GVHD rates ranged from ~25% to ~40% and the chronic GVHD incidence ranged from ~25% to 40%.

Another Chinese study of 29 patients with a median age of 17 (range 14–30) years showed 1-year survival of 91.7% [70]. They used conditioning with Cy, 25 mg/kg/day on days −4 and −3; Bu, 3.2 mg/kg/day on days −6 and −5; Flu, 40 mg/m²/day on days −6 to −2 and GVHD prevention with Cy, 50 mg/day on days +3 and +4 and short MTX and long CSP through 1 year.

Yet another Chinese trial [71] reported outcomes on 29 patients given HLA-haploidentical grafts at three centers. Their conditioning regimen consisted of ATG/Bu/low-dose CY, and Flu. GVHD prevention included CY on days + 3 and +4, followed by MMF and CSP. Their acute GVHD incidence was 25.9% with 7.4% grades III and IV GVHD. One-year failure free survival was 96%.

While results of the Chinese trials were impressive, the wisdom of using Bu in patients with aplastic anemia could be questioned. Bu is exceptionally good at ablating marrow. However, a marrow-ablative drug is not needed in aplastic anemia patients since their marrows are empty to start with. Equally importantly, Bu is a poor immunosuppressive agent; however, as we have seen, immunosuppressive properties are uniquely important for conditioning aplastic anemia patients for transplantation. Taken together, Bu’s strengths and weaknesses make the drug a less than ideal conditioning choice for patients for patients with aplastic anemia undergoing allogeneic hematopoietic cell transplantation.

7. Conclusion

Early attempts at marrow transplantation for severe aplastic anemia in the 1950s and early 1960s, before a full understanding of transplantation biology was obtained, were a complete failure. Years of subsequent, systematic animal experimentation laid the groundwork for a resumption of human marrow transplantation trials in 1970. Marrow donors in these early transplantations were in all cases HLA-identical siblings. Graft rejection and GVHD were the major hurdles encountered, and survivals did not exceed 50%. Further systematic animal experimentation dissected these problems and found ways to overcome them. Subsequent clinical translation of the advances achieved in preclinical models resulted in stepwise improvements in HLA-identical sibling transplantation for aplastic anemia, with current survivals approaching 100%.

Rapid developments in the understanding of the MHC including molecular HLA typing and the development of unrelated volunteer donor registries, have led to the routine use of marrow grafts from HLA-matched unrelated donors with increasing success. Cooperation between the various international donor registries has enabled access to HLA data of more than 40 million unrelated volunteers. However, identifying HLA-matched unrelated volunteer donors for patients from minority populations has remained a problem.

Success in overcoming the donor problem for minority patients has come from animal experimentation, which led to the successful clinical translation of highly effective conditioning regimens and GVHD prevention protocols for HLA-haploidentical, related hematopoietic cell transplantation. This breakthrough has enabled finding suitable donors for nearly all patients.

The current priority order of donor source for marrow transplantation is: 1. HLA-identical sibling; 2. HLA-matched unrelated donor; and 3. HLA-haploidentical related donor. Marrow transplantation from each of these donor sources may be preferable to non-transplant IST. Implementation of this first-line treatment strategy will provide patients with severe aplastic anemia the best chance of long-term disease-free survival.