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Keywords:

  • cord blood transplantation;
  • HSC transplantation;
  • engraftment kinetics

Summary

  1. Top of page
  2. Summary
  3. 2009 general perspective of unrelated UCBT as a modality of HSCT: pros and cons and the risk of engraftment failure
  4. Clinical study of ‘Dual UCB/TPD–MHSC Transplants’: a strategy to increase likelihood of UCBT engraftment with single units of relatively low cell content and HLA mismatched
  5. How do ‘dual transplants’ compare to double UCB transplants?
  6. Use of other TPD cells in patients recipients of ‘dual transplants’
  7. Future perspectives
  8. Acknowledgements
  9. Funding
  10. References

We developed the strategy of umbilical cord blood transplants (UCBT) with co-infusion of a limited number of highly purified mobilized haematopoietic stem cells (MHSC) from a human leucocyte antigen (HLA) unrestricted third party donor (TPD). Short post-transplant periods of neutropenia were usually observed in adults with haematological neoplasms receiving UCBT with a relatively low cell content and 0–3 HLA mismatches after myeloablative conditioning. This resulted from an early and initially predominant engraftment of the TPD–MHSC. After a variable period of double complete TPD + UCB chimerism, final full UCB chimerism was achieved (cumulative incidence >90%) within 100 d. Early recovery of the circulating neutrophils resulting from the ‘bridge transplant’ of the TPD–MHSC reduced the incidence of serious neutropenia-related infections, also facilitating the use of drugs with myelosuppressive side effects to combat other infections. The observed incidence of graft-versus-host disease and relapses was low, with overall and disease-free survival curves comparable to those of HLA identical sibling transplants. Post-transplant recovery of natural killer cells occurred soon after the transplant and B cells recovered around 6 months, but T-cell recovery took more than 1 year. Available data show that T cell recovery derives from UCB–HSC through thymic differentiation and that cytomegalovirus (CMV)-specific lymphocytes develop following CMV reactivations.

The main objective of haematopoietic stem cell transplantation (HSCT) is to reconstitute all or part of a recipient’s haematopoietic system (HS), when it has been damaged by disease or previous therapeutic interventions. Currently, diseases that may benefit from HSCT include: (i) metabolic diseases (Sir Archibald Garrod’s ‘congenital metabolic errors’) that disturb the normal development and functionality of the HS, either as a whole or in any of its functional divisions, myeloid or lymphoid; (ii) acquired bone marrow failure syndromes, toxic or autoimmune; (iii) other acquired autoimmune and immune deficiency diseases; (iv) tumours arising from the HS; and (v) other tumours whose treatment may imply serious damage to the HS or that may benefit from immunotherapeutic effects.

In contrast to other transplants, HSCT involves the interplay of two genetically different immune systems that need to be modulated for mutual tolerance. The recipient’s immune system must be modulated before the transplant to avoid rejection. This can be accomplished with radiation and drugs, which may produce undesirable toxic damage to different organs and tissues, most importantly the HS, the gastrointestinal tract, liver and lungs. In turn, the allo-reactivity of the immune system derived from the graft has to be modulated to avoid severe damage to host tissues and organs, i.e., graft-versus-host disease (GVHD). This may require pre-implant manipulations of the transplantation product or post-transplant actions involving use of drugs or the infusion of selected donor cell populations. Modulation of the graft allo-reactivity may have adverse consequences: reduction or anti-rejection effects, interference with the development of adaptive immunity against pathogens and loss of reactivity against tumour cells (graft-versus tumour; GVT). This is an important therapeutic factor for HSCT used to treat neoplasms because it may eradicate tumour stem cells, but is only effective in the face of residual tumour loads. Because of this, treatment of patients with neoplasms requires that the pre-transplant conditioning regimens include doses of radiation or drugs capable of destroying tumour cells; this, however, also increases the procedure’s toxicity. Reduced-intensity conditioning regimens (RIC) seek to minimize undesirable toxic effects, both to bone marrow and other tissues and organs, but for treatment of primary myeloproliferative diseases (MPD) the margin to reduce marrow toxicity may be limited by possible loss of anti-tumour effect.

HSCT is therefore not a homogeneous procedure, but may have diverse designs with important differences based on factors such as: (i) indications and recipients’ condition; (ii) donors; (iii) source of the transplant product; (iv) conditioning regimen; and (v) post-transplant immunomodulation. As a transplant material, umbilical cord blood (UCB) has, aside of its limited quantity, the advantages of containing a high proportion of cells with very high proliferative potential, and lymphoid cells that mainly consist of antigen-inexperienced (naïve) and CD4+ Cd25+ T-reg cells (Broxmeyer et al, 1989; Payne et al, 1995; D’Arena et al, 1998). Related to these, late engraftment, low severity of GVHD despite human leucocyte antigen (HLA) mismatch and late reconstitution of adaptive immunity are distinctive features of UCB transplantation (UCBT). This article reviews the use of cells from voluntary third party donors (TPD) to improve the outcome of UCBT in adults, within the context of other possible approaches.

2009 general perspective of unrelated UCBT as a modality of HSCT: pros and cons and the risk of engraftment failure

  1. Top of page
  2. Summary
  3. 2009 general perspective of unrelated UCBT as a modality of HSCT: pros and cons and the risk of engraftment failure
  4. Clinical study of ‘Dual UCB/TPD–MHSC Transplants’: a strategy to increase likelihood of UCBT engraftment with single units of relatively low cell content and HLA mismatched
  5. How do ‘dual transplants’ compare to double UCB transplants?
  6. Use of other TPD cells in patients recipients of ‘dual transplants’
  7. Future perspectives
  8. Acknowledgements
  9. Funding
  10. References

In the two decades since UCBT was introduced to clinical practice, (Gluckman et al, 1989) this HSCT modality has evolved into an effective option for the treatment of a number of haematological malignancies and many non-malignant disorders requiring haematopoietic reconstitution for patients lacking a readily available related or unrelated volunteer donor with adequate HLA compatibility (Rubinstein et al, 1998; Rocha et al, 2000; Schoemans et al, 2006). Other applications of UCB stem cells in the field of reparative medicine are now envisioned and are the focus of ongoing active basic and translational research (Friedlander et al, 2007; Goldberg et al, 2007; Sueblinvong et al, 2007; Harris, 2008; Park et al, 2008).

Recognized advantages of UCBT for haematological reconstitution include easy procurement without risks for the donor, reduced risk of transmitting infections, fast accessibility and relatively low incidence and severity of GVHD despite one to three out of six HLA mismatches. At least one UCB unit with a minimum of four out of six A-B-DR HLA-matches (antigenic for loci A and B and allelic for locus DR) may now be found for the great majority of patients in the worldwide network of UCB banks. On the other hand, the main disadvantages of unrelated UCBT transplantation are the limited number of HSC contained in the amount of UCB that can be collected after birth, the possibility of transmitting undetected haematological pathologies and the unavailability of the donor for additional donations (Barker et al, 2002; Wagner et al, 2002; Brunstein et al, 2007a; Hwang et al, 2007; Kurtzberg et al, 2008; Prasad et al, 2008).

Several registry and single institution studies have shown that, for treatment of both paediatric and adult patients with acute leukaemia, UCBT may result in overall survival (OS) and disease-free survival (DFS) rates similar to those achieved with transplants from voluntary donors, although reported series of UCBT generally include higher proportions of patients at higher risk. This is usually a consequence of a tendency to use UCBT as a last resort, and also because the shorter waiting time may be a factor in selecting UCBT patients whose diseases may be prone to relapse quickly. Slow and low rates of engraftment, infections during lengthy post-transplant neutropenia and toxicity of preparative regimens have been recognized as factors for early transplant-related mortality of UCBT in large registry-based observational studies (Rocha et al, 2001; Rocha et al, 2004; Eapen et al, 2007; Sanz, 2004; Takahashi et al, 2004, 2007a; Laughlin et al, 2004; Tse et al, 2008). Low cell content in the transplanted product and a higher degree of HLA incompatibility are, in turn, the main factors for graft failure and late engraftment (Rubinstein et al, 1998; Wagner et al, 2002). All of these have limited the wider use of UCBT, especially in adults. Another concern with UCBT relates to the possibility of poor development of anti-tumour and adaptive immunity against pathogens. Obviously, a high rate of engraftment is a necessary pre-requisite for an adequate evaluation of UCBT immune-related GVHD, GVT and development of adaptive immunity. Strategies that have been pursued to obtain high rates of engraftment include, among others, ex-vivo expansion of HSC of an aliquot of the transplanted UCB, intraosseous infusion, infusion of two UCB units, and the co-infusion of TPD MHSC (proposed by our group). All these may be combined with RIC.

Over the past decade, several clinical experiments with expanded cord blood cells from an aliquot of a single unit UCBT did not result in clinical benefit and, in the absence of markers to trace the progeny of the expanded cells, no evidence of a significant contribution of the culture-expanded cells to engraftment was produced (Kögler et al, 1999; Shpall et al, 2002; Jaroscak et al, 2003; de Lima et al, 2008). In fact, some of these procedures could imply reduction of the engraftment potential of the transplanted CB unit because of detraction of native HSC. We performed animal studies in the severe combined immunodeficiency non-obese diabetic (SCID-NOD) mouse model to compare engraftment capacity of native and ex vivo expanded CB CD34+ under different culture conditions. Results suggested that, although ex vivo expansion of CB cells preserved the long-term repopulating ability of the sample, delayed engraftment was associated with the transplantation of these manipulated cells in the xeno-transplant model used (Güenechea et al, 1999).

Based on the well known fast engraftment characteristic of mobilized HSC transplants (MHSCT) and the likewise rapid engraftment and lack of GVHD observed in patients receiving mega-doses of highly T cell-depleted MHSCT from haploidentical donors (Aversa et al, 1998), we hypothesized that co-infusion of a certain number of MHSC from a haploidentical donor could result in early recovery of the absolute neutrophil count (ANC), thus providing support for the engraftment of UCB cells. To test the capacity for competitive engraftment of these two sources of HSC, a fixed number of positively selected CD34+ cells from UCB and different numbers of CD34+ MHSC were co-infused to SCID-NOD mice. Our data showed greater potential of human UCB cells for competitive sustained engraftment in the xenogeneic model used that was only abrogated by the co-infusion of very large numbers of MHSC cells (Ramírez et al, 2005).

On the clinical side, Fernández et al (2000, 2001) first assayed double UCBT, simultaneously infusing the best available UCB unit immediately after thawing and the cells resulting from the ex vivo expansion of CD34+ cells recovered, by positive selection, from the entire second best available UCB unit. Expansion cultures were performed using two different cytokine cocktails in the culture medium. Assessment of chimerism by means of DNA analysis of the discriminating HLA alleles showed that none of the five patients transplanted using this approach experienced significant contribution of the expanded cells to the recovery of circulating granulocytes. On the contrary, short periods of post-transplant neutropenia were observed in the three patients who received, as compassionate treatment, a co-infusion of a single UCB unit and a relatively small number of mobilized CD34+ cells obtained from a haploidentical donor by positive selection, containing <104/kg CD3+ cells. The prompt recovery of the ANC was the result of the early and transient engraftment of the haploidentical MHSC (Fernández et al, 2000, 2001).

Clinical study of ‘Dual UCB/TPD–MHSC Transplants’: a strategy to increase likelihood of UCBT engraftment with single units of relatively low cell content and HLA mismatched

  1. Top of page
  2. Summary
  3. 2009 general perspective of unrelated UCBT as a modality of HSCT: pros and cons and the risk of engraftment failure
  4. Clinical study of ‘Dual UCB/TPD–MHSC Transplants’: a strategy to increase likelihood of UCBT engraftment with single units of relatively low cell content and HLA mismatched
  5. How do ‘dual transplants’ compare to double UCB transplants?
  6. Use of other TPD cells in patients recipients of ‘dual transplants’
  7. Future perspectives
  8. Acknowledgements
  9. Funding
  10. References

Protocol design

Following this initial observation a phase I-II pivotal study was designed to further evaluate the dual UCB/TPD–MHSC transplant (‘dual transplant’) approach in patients with high-risk haematological neoplasms. Progress reports of this study were presented after the inclusion of 11, 27 and 55 patients (Fernández et al, 2003; Magro et al, 2006; Bautista et al, 2009). The first three patients received our then usual myeloablative conditioning regimen consisting of total body irradiation (TBI), 1200 cGy delivered in six 200 fractions twice a day (lungs shielded at 800 cGy) in 3 d, followed by cyclophosphamide (CTX) 60 mg/kg per d × 2 d and anti-thymocyte globulin (ATG). All other patients underwent regimens of lower extra-haematological toxicity based on the use of fludarabine (total dose of 120 mg/m2 given in 2 d), TBI, 1000 gGy delivered in five 200 fractions in 3 d (lungs also shielded at 800 cGy), and the same doses of CTX. This regimen was preferentially used in patients with lymphoid malignancies. Patients with myeloid neoplasms, or contraindications to TBI, received busulfan (4 mg/d × 2 d, up to year 2006) or Busilvex (3·2 mg/kg × 2 d, from year 2006). Up to the year 2000, the ATG used was the equine Linfoglobulina (Sangstat, Lyon, France) at a dose of 30 mg/kg given 1 d before transplant. After 2000, the ATG used was rabbit Timoglobulina (Genzyme, Mercy l’Etoile, France), given at a dose of 2 mg/kg on days -2 and -1 before the transplant. Two patients with lymphoma in unstable remission had cytarabine (2000 mg/m2 per d × 5 d) added to their conditioning regimen. All patients received granulocyte colony-stimulating factor, started after the transplant at a dose of 30 MU/d and continued until a sustained ANC >0·5 × 109/l was reached. The dose was then adjusted as needed to maintain the ANC above this level. To intensify the immunosuppressive conditioning, and as GVHD prophylaxis, patients received ciclosporin A from day -5 at a dose of 3 mg/kg daily, adjusted to a serum level of 180–250 ng/ml. It was administered orally as soon as oral tolerance developed, and was continued until full UCB engraftment was achieved, at which point tapering was immediately initiated if there were no signs of GVHD. Methylprednisolone was given i.v. at a dose of 1 mg/kg per d from day −2 to days +10 to +14, and was rapidly tapered thereafter unless there was a clinical indication for its maintenance. Corticosteroid-based treatment (methylprednisolone, 1–2 mg/kg per d) was given when grade II or higher acute GVHD (aGVHD) developed.

Patients and donors

Patients included in our study of ‘dual transplant’ were 34 males and 21 females with a median age of 34 years (range 16–60) and a median weight of 70 kg (range 43–95), 89% serologically positive for CMV. The underlying disease was high risk MPD in 27 and high risk lymphoproliferative disease (LPD) in 28 (Bautista et al, 2009).

The transplanted UCB units had 0–3 HLA mismatches (antigenic for loci A and B, allelic for DR) both in the direction of GVHD and rejection; the great majority had 1–2 mismatches and their cellularity was relatively low: median total nucleated cells (TNC) of 2·39 × 107/kg (range 1·14–4·30) and median CD34+ cells of 0·11 × 106/kg (range 0·035–0·37). The TPD donor was the mother for four, another haploidentical relative for 34 (62%) and an individual without shared haplotypes for 17 (31%). The median number of the infused TPD MHSC, positively selected as CD34+ and/or CD133+ cells, was 2·4 × 106/kg (range 1·05–3·34), median number of CD3+ cells being 3·2 × 103/kg with a range of 0·5–15·6, only one patient receiving >104/kg of these (Bautista et al, 2009).

Engraftment

We initially selected the TPD from the patient’s HLA haploidentical relatives. Failure of engraftment of the TPD MHSC cells was observed in four of the initial 11 patients whose mothers were used as TPD. Because of this observation, mothers were no longer used as TPD. This lack of engraftment of mothers’ MHSC could not be explained by intrauterine sensitization of the recipients to the non-inherited maternal antigens (NIMA), as these were present in the MHSC cells from brothers and sisters used as TPD, which in all cases engrafted. Nevertheless, sensitization to maternal minor compatibility antigens is a possibility (Mommaas et al, 2005).

One patient had early rejection of the initially transplanted UCB unit; in two others there was primary failure of engraftment of the UCBT related to the lack of viable progenitor cells in the infused UCB product, as ascertained by the control CD34+ and CFU assays in the thawed products. In all cases, patients maintained an ANC derived from the engrafted TPD sufficiently high as to remain infection-free until the engraftment of a second UCB unit, given after intervals of 32, 34 and 63 d. When data on the first of these patients (Fig 1) were revised, it was discovered that an error had been made in the selection of the TPD, who happened to be an entirely different HLA sibling (Magro et al, 2006). Following this unexpected observation, non-haploidentical TPD have been used whenever a suitable haploidentical donor was not available. No difference was observed in the pattern of engraftment of the TPD of the haplo and non-haplo identical TPD (34 vs. 17). In addition, selection of the TPD–MHSC as CD34+ or as CD133+ cells did not result in significant differences in the post-transplant recovery of granulocytes or platelets.

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Figure 1.  Post transplant course of engraftment and chimerism of a ‘dual transplant’ whose initial UCB transplant lacked HSC and whose TPD did not share any HLA haplotype. The patient was a 28-year-old male, who first received third-party donor (TPD) cells from a sister, with whom he did not share any haplotype, and a cord blood (CB) unit that proved to lack viable progenitor cells as ascertained by post-thaw CD34+ counting and colony-forming unit control cultures. Absolute neutrophil count (ANC) recovery and full TPD chimerism were detected from day +10. On day +23 the bone marrow was morphologically normal, although there was no evidence of CB engraftment. At that time he also acheived transfusion independence, with lymphocyte counts remaining low (<0·5 × 109/l). He had no manifestations of graft-versus-host disease (GVHD) and no evidence of CB engraftment had been detected by day +30. He then received fludarabine (30 mg/kg × 3 d) and antithymocyte globulin (30 mg/kg × 3 d) followed by a second CB unit on day +34. Following this he continually maintained normal blood counts and bone marrow cellularity. Progressive replacement of the TPD cells by cells from the second CB unit was documented from day +15 after the second CBT. Time to CB-ANC >0·5 × 109/l was 49 d, and time to full CB chimerism 70 d from the second CBT. The patient is alive and well more than 7 years from the transplant (Magro et al, 2006).

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Cumulative curves of ANC and UCB–ANC recovery, as well as the cumulative curves of achievement of full UCB chimerims, are shown in Fig 2 (Bautista et al, 2009). Time to ANC >0·5 × 109/l ranged from 9 to 36 d (P50 = 10, P75 = 14) with a maximum cumulative incidence (MCI) of 0·96 (95% confidence interval (CI): 0·91–1·00). It was 100% among patients receiving MHSC from non-maternal TPD. The estimated time to UCB–ANC >0·5 × 109/l ranged from 12 to 57 d (P50 = 21, P95 = 57 and MCI=0·95 (95% CI: 0·89–1·00). Final full UCB chimerism was achieved in 50 patients, representing a MCI of 0·91 (95% CI: 0·84–0·99), after transient periods of double UCB and TPD chimerism. Time to full UCB chimerism ranged from 11 to 186 d (P50 = 44, P90 = 97). Median times to sustained platelet counts >20 × 109/l and >50 × 109/l were 32 and 57 d, with MCIs of 0·78 (95% CI: 0·68–0·90) and 0·69 (95% CI: 0·58–0·82) respectively (Bautista et al, 2009).

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Figure 2.  Cumulative curves of engraftment of recipients of ‘dual transplants’. ANC-500: recovery of sustained of ANC to >0·5 × 109/l. Range 9–36 d; P50 = 10, P75 = 14 and P95 = 31 d; MCI 0·96. CB ANC-500: recovery of ANC of Cord Blood origin to >0·5 × 109/l. Range 12–57 d, P50 = 21 and P95 = 57 d; MCI 0·95. Full CB chimerism: range 11–186 d, P50 = 44 and P95 = 97 d; MCI 0·91) Bautista et al, 2009.

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Data on the sequential evaluation of chimerism showed initial predominance of TPD DNA both in granulocytes and mononuclear circulating and marrow cells, followed by progressive replacement by cells from the UCBT (Fig 3) (Magro et al, 2006). The effacement of the TPD cells could be the result of competitive replacement, although immune rejection by the engrafting UCB could be involved. Results of mixed lymphocyte culture performed with post-transplant and TPD lymphocytes were not informative in this regard (S. Querol, unpublished data).

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Figure 3.  Progression of CB engraftment in 12 adults recipients of ‘dual transplants’. Proportions over time of CB DNA in circulating granulocytes (bsl00072) and mononuclear cells (•) (Magro et al, 2006).

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The early recovery of the ANC that results from the ‘bridge’ engraftment of the TPD MHSC provides not only protection against bacterial and fungal infections, but also the capacity to withstand treatments with gancyclovir and other antimicrobials with myelosuppressive side effects.

Our data clearly showed that the ‘dual transplant’ strategy makes achievement of engraftment and full UCB chimerism feasible in almost any patient with 0–3 HLA mismatched units and cell content significantly lower than the thresholds of 2·5 × 107/kg TNC and 0·17 × 106/kg CD34+ cells that are usually quoted as required for a high probability of engraftment (Wagner et al, 2002; Brunstein et al, 2007a). Units of such size are rarely available for high weight patients. This strategy makes it feasible to prioritize HLA match on cell content in the selection of the UCB to be used when a higher match may considered advantageous. Our results were recently confirmed by another group in a series of poor risk patients (Rich et al, 2008).

Survival, GVHD, relapses and infections

Figure 4 shows the OS and DFS curves of the 55 patients included in our study of ‘dual transplants’. These survival data compare favourably with those of patients receiving HSC transplants from HLA identical siblings in our centre (Fig 5).

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Figure 4.  Kaplan–Meier Survival curves of 55 adults with poor risk haematological neoplasms, recipients of ‘dual transplants’. Curves of patients <40 and >40 years without significant differences (P = 0·06). Upper panel, Overall Survival. Lower panel, Disease Free Survival (Bautista et al, 2009).

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Figure 5.  Survival curves of poor risk leukaemia adult patients treated with ‘dual transplants’ or HSCT from HLA identical siblings. Upper panel, Overall survival curves (54 vs. 34 patients); Lower panel, Disease Free Survival (48 vs. 40 patients). Solid lines: Dual Transplants. Dotted lines: HSCT from HLA identical siblings. No significant differences in OS and DFS of the two groups.

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Incidences of serious acute and chronic GVHD were both low. Cumulative incidence of grades I-IV, II-IV and IV aGVHD were 0·63, 0·28 and 0·11 respectively. Chimerism analysis of DNA extracted from skin biopsies of GVHD showed the presence of DNA from the UCBT in several cases but DNA from the TPD was not detected in any (Bautista et al, 2009). GVHD was the primary cause of death in three patients. All others responded to treatment; two of these required infusion or mesenchymal stem cells (MSC) obtained from the TPD. The observed relapse rate was relatively low (cumulative incidence of 0·17, CI: 0·08-0·35) despite the high-risk nature of the transplanted patients, suggesting an important GVT effect despite the low incidence of GVHD (Bautista et al, 2009). All these data compared favourably with data on other HSCT modalities.

The incidence of serious neutropenia-related infections was unremarkable. The main cause of morbidity and mortality was post-engraftment opportunistic infection; polyomavirus and cytomegalovirus (CMV) reactivations were the most frequently observed, denoting slow development of adaptive immunity. Nevertheless, the incidence of these complications decreased after the fourth month, suggesting development of some degree of specific immune capacity. One patient developed Epstein–Barr virus (EBV)-related LPD that ultimately caused her death (Bautista et al, 2009).

Immune reconstitution

Recipients of UCBT have been reported to have long-lasting post-transplant periods of deficient adaptive immunity, with fully functional natural killer (NK) cells recovering earlier than B and T cells (Moretta et al, 2001; Szabolcs & Niedzwiecki, 2007; Beziat et al, 2009; Martín-Donaire et al, 2009). This pattern of early NK and late T-cell recovery is consistent with the clinical observation of high GVT effect and low incidence of serious GVHD, because the NK cells are being recognized as important effectors of GVT. We have observed this same pattern in adult recipients of ‘dual transplants’ (Martín-Donaire et al, 2009) Most of our patients recovered normal numbers of circulating NK cells within the initial 3 months after the transplant, implying that, in a large proportion of cases, this occurred while the patient still was in double UCB/MHSC chimerism. Normal levels of circulating B and T cells were achieved after longer intervals, when the patients were in full UCB chimerism, in both granulocytes and mononuclear cells. The majority of patients reached normal levels of B cells by month six; T-cell populations required more than 1 year to recover in the great majority of the patients (Fig 6) (Martín-Donaire et al, 2009). At the time of writing, analyses of chimerism performed in isolated subpopulations in the early post-transplant period are pending, but it is conceivable that the TPD–MHSC might contribute to the development of the circulating NK cells in this early period. This could have interesting implications regarding the selection of the TPD. According to what is known about the anti-leukemic and anti-GVHD effects of donor NK cells in recipients of haploidentical HSCT and UCBT from donors who are killer cell immunoglobulin-like receptor (KIR) incompatible with the recipient (Ruggeri et al, 2005; Grzywacz et al, 2008; Willemze et al, 2009), the idea of selecting TPD who are both KIR compatible with the transplanted CB unit and KIR incompatible with the recipient for ‘dual transplants’ is appealing.

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Figure 6.  Cumulative incidence of recovery of lymphocyte subpopulations in recipients of ‘dual transplants’. Upper panel: NK, B and T cells. Lower panel: T cell CD4+ and CD8+ subpopulations. The curves represent the cumulative incidence of recipients of ‘dual UCB/TPD–MHSC transplants’ reaching circulating absolute numbers of different cell sub-populations ≥ the median values of 22 normal controls (Martín-Donaire et al, 2009).

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The observed late initiation of T-cell recovery in our patients does not support an initial predominant contribution of the expansion of the naive T cells infused with the UCBT. This may be, at least to a certain extent, a consequence of the purging effect of the ATG given as part of the conditioning (Parkman, 2008). Output of thymic cells can be evaluated by the quantitation of signal joint T-cell receptor rearrangement excision circle (sjTREC)-bearing cells (Gill et al, 2003; Junge et al, 2007; Kilpatrick et al, 2008). Reports on sjTRECs after UCBT in adults are few and somewhat discrepant (Talvensaari et al, 2002; Komanduri et al, 2007). Analyses of the sjTRECs-bearing cells within subpopulations of lymphocytes of naive and committed immunophenotypes at different intervals following transplant in recipients of ‘dual transplants’, suggest that adults receiving UCBT according to our protocol maintain, when compared to normal controls (adults, children and UCB samples), a substantial degree of thymic function that appears to be an important factor for the recovery of the T-cell populations. In addition, our data on the course of recovery of cells of naive, effector and memory phenotypes within the CD3+ CD4+ and CD3+ CD8+ subpopulations are consistent with expansion of naive ‘recent thymic emigrates’ (RTE) generated in the recipient’s thymus from the UCB–HSC. This could result from the combined effects of the post-transplant lymphopenic environment (homeostatic expansion) and exposure to antigenic stimuli. Possibly, this could explain the observed lagging recovery of the absolute numbers of circulating cells of naïve phenotype relative to the faster recovery of the committed (effector and memory) subsets, despite a sustained thymic cell output that, estimated on the basis of levels of sjTRECs, is not poorer than in normal infantile and adult controls (Martín-Donaire et al, 2009). But, although levels of sjTRECs in circulating T cells populations beyond the third month post-transplant were significantly high compared to different normal controls (adults, 6- to 24-month-old children and UCB samples), the efficiency of the whole process of generation of committed antigen-specific T lymphocytes appears to be poor. It is conceivable that manoeuvres that activate thymic development of the T-cells populations might be valuable for enhancing the recovery of adaptive immunity in recipients of ‘dual transplants’, although enhancement of GVHD might be an associated risk. Development of UCB-derived CMV-specific cytotoxic T lymphocytes (CTL) was observed in most of our patients with positive CMV serology prior to the transplant who developed CMV reactivations (Martín-Donaire et al, 2009). Development of antigen-specific T-lymphocyte immunity to different herpesviruses in recipients of UCBT has been previously described, and correlated with decreased leukaemia relapses (Cohen et al, 2006; Parkman et al, 2006). It is thus conceivable that recipients of UCBT could also benefit from appropriately designed manoeuvres for post-transplant active vaccinations.

How do ‘dual transplants’ compare to double UCB transplants?

  1. Top of page
  2. Summary
  3. 2009 general perspective of unrelated UCBT as a modality of HSCT: pros and cons and the risk of engraftment failure
  4. Clinical study of ‘Dual UCB/TPD–MHSC Transplants’: a strategy to increase likelihood of UCBT engraftment with single units of relatively low cell content and HLA mismatched
  5. How do ‘dual transplants’ compare to double UCB transplants?
  6. Use of other TPD cells in patients recipients of ‘dual transplants’
  7. Future perspectives
  8. Acknowledgements
  9. Funding
  10. References

In our centre we do not have experience regarding the transplant of two unmanipulated cord blood units, a procedure pioneered by the Minnesota group, and now used by several groups, as a strategy to improve engraftment and overall results of UCBT, often combined with submyeloablative conditioning, to transplant patients of advanced age or with co-morbidities (Barker et al, 2005; Ballen et al, 2007; Brunstein et al, 2007b; Chen & Spitzer, 2008). Representative of this approach are the results reported in a series of 110 patients transplanted after submyeloablative conditioning, (93 with two units, 17 with single units of high cell content) (Brunstein et al, 2007b). Recovery of ANC > 0·5 × 109/l was achieved by 92% of the patients after intervals of 0–32 d (P50 = 12 and P75 = 21), with bone marrow chimerism of 8–100% (median, 89%) at day 21 and 34–100% (median, 100%) at day 100. Although in all patients receiving two UCB units only one unit contributed to long-term haematopoiesis, contributions of both units to the marrow chimerism was detected in 43% of patients at day 21, 9% at day 100 and 0% at 1 year. Although not specified, on the basis of these data, it may be presumed that autologous cells initially contributed to the recovery of the ANC in patients with short periods of post-transplant neutropenia. At 3 years the incidence of disease relapse or progression was 31% (95%CI 21–41%) and the probability of event-free survival was 38% (95%CI, 28–48%). Results were better in those receiving two UCB units versus one: 39% (27–51%) vs. 24% (4–44%) (P = 0·05). According to the results of this and other studies (Majhail et al, 2008), the short term benefit of using two UCB units compared to single unit UCBT seems to be a lower risk of primary graft failure, rather than earlier engraftment; autologous reconstitution after RIC seems to be important factor in patients with prompt recovery of the ANC.

It is worth noting that the use of different criteria to evaluate the cellularity of transplanted UCB units makes it difficult to compare the data reported by different transplant centres (Regidor et al, 2008). In any event, the incidence of relapses, serious GVHD and survival (both OS and DFS) of double UCBT do not appear to be better than those observed in our patients receiving ‘dual transplants’. These, in turn, are not worse than those in our patients receiving bone marrow or MHSC-transplants from HLA identical family donors (M.N. Fernández, A. La Iglesia, A. Sebrango, I. Vicuña, E. Ojeda, G. Bautista, C. Regidor, R. Cabrera, unpublished observations) (Fig 6). Regarding costs, at least in Spain, the cost of obtaining the TPD–MHSC is less than one third the amount charged by UCB banks for one UCB unit.

Recent findings could open new perspectives on double UCBT. Co-infusion of an unmanipulated CB unit and cells from a second one after ex-vivo expansion using novel culture methods, in media containing recombinant cytokines and an engineered form of the Notch ligand, have resulted in accelerated myeloid engraftment of CB HSC expanded cells (Delaney et al, 2008). The relative contribution of the expanded and non-cultured grafts over time was determined through DNA-based chimerism assays. These analyses showed that the expanded cells resulted in earlier, but not permanent, myeloid engraftment, suggesting provision of short term repopulating cells. The most appealing aspect was that the same investigators also reported results of preclinical assays that could suggest that cells expanded with this new method could also have the capacity for accelerated T cell recovery as well as to facilitate myeloid and lymphoid engraftment of the non-cultured CB cells (Dallas et al, 2008a,b). This opens the possibility of using HSC from CB units of low cell content, usually discarded for CB banking, to produce cell products, that might be available ‘off the shelf’, to enhance myeloid and lymphoid reconstitutions of CB transplants (Querol, S, Anthony Nolan Research Institute, London, UK, personal communication).

Use of other TPD cells in patients recipients of ‘dual transplants’

  1. Top of page
  2. Summary
  3. 2009 general perspective of unrelated UCBT as a modality of HSCT: pros and cons and the risk of engraftment failure
  4. Clinical study of ‘Dual UCB/TPD–MHSC Transplants’: a strategy to increase likelihood of UCBT engraftment with single units of relatively low cell content and HLA mismatched
  5. How do ‘dual transplants’ compare to double UCB transplants?
  6. Use of other TPD cells in patients recipients of ‘dual transplants’
  7. Future perspectives
  8. Acknowledgements
  9. Funding
  10. References

One of the disadvantages of UCBT is the unavailability of the donor for additional donations. This could be overcome by using different cell types from TPDs for different cell therapy actions. We have infused cells other than MHSC from TPD to recipients of ‘dual transplants’ with different objectives. Donor cells that may have the capacity of producing preventive or therapeutic effects include MSC, NK cells, pathogen or tumour-specific CTL and T-reg lymphocytes.

MSC

In preclinical models MSC have been shown to have immunosuppressive properties, delaying skin graft rejection, and inconsistent results have been reported regarding prevention and treatment of GVHD (Sudres et al, 2006; Tisato et al, 2007). In the clinical setting, facilitation of engraftment and favourable effects of GVHD have been reported by the infusion of donor MSC to HSCT recipients (Le Blanc et al, 2004, 2007; Lazarus et al, 2005; Ringdén et al, 2006; Ball et al, 2007) although a higher incidence of relapses could be a risk (Ning et al, 2008; Vianello & Dazzi, 2008).

MSC obtained by means of cultures under good manufacturing practice conditions of bone marrow samples from the same TPD were given to nine recipients of ‘dual transplants’ in a phase I-II open label exploratory clinical study to evaluate tolerance and possible favourable effects on engraftment, development of GVHD or any other effects on immune reconstitution (Gonzalo-Daganzo et al, 2009) We did not observe any immediate adverse effects and no significant differences were found regarding ANC recovery, UCB engraftment or incidence of aGVHD grade >I, compared to patients who did not receive MSC, although none of the nine patients developed grade III–IV aGVHD. On the other hand, a remarkable therapeutic effect resulted from the infusion of additional doses of MSC to two patients who developed Grade II GVHD refractory to steroids. On the basis of these results (and results in other HSCT recipients also receiving MSC for refractory GVHD) we discontinued the prophylactic use of the expensive MSC products and are now evaluating its use as preemptive treatment for aGVHD grade >I in recipients of HSC transplants.

NK cells

NK cells, functionally regulated through activating and inhibitory receptors, appear to play a significant role in reducing GVHD and eradicating residual disease after HSCT. These actions are the result of cytotoxic effects on recipient antigen-presenting cells and leukaemic cells when the NK cells are not inhibited by ligands to the KIR and other receptors that recognize class I HLA molecules of the recipient. Peripheral blood NK cells (CD3-depleted lymphopheresis products) have also been used for adoptive immunotherapy in chemotherapy-refractory patients (Ruggeri et al, 2005; Grzywacz et al, 2008).

One of our patients with acute myeloid leukaemia suffered a relapse after a ‘dual transplant’ that proved refractory to several courses of combined chemotherapy. It was found that her entirely HLA mismatched TPD was KIR compatible with the transplanted UCB unit and KIR incompatible with the patient. A complete remission was obtained after a course of fludarabine and ATG, followed by the infusion of new doses of TPD–MHSC and two consecutive infusions, given 1 week apart, of peripheral blood selected NK cells obtained from the TPD (Fig 7) This observation is consistent with the reported antikeukemic effects of KIR-incompatible transplants (Ruggeri et al, 2005; Willemze et al, 2009) and supports the selection of TPD for ‘dual transplants’ on the basis of KIR compatibility with the UCB and incompatibility with the recipient. Additional clinical assays, properly designed to evaluate this possibility, are desirable.

image

Figure 7.  Chemotherapy refractory relapsed AML after a ‘dual transplant’ responding to infusions of NK cells from the TPD. The TPD was the patient’s husband, who was KIR incompatible with her and KIR compatible with the transplanted UCB unit. After re-conditioning with fludarabine and ATG the patient received both T-cell depleted MHSC and selected NK cells from her husband. This was followed by recovery of the ANC and bone marrow cellularity with mixed UCB/TPD chimerism. A second infusion of NK cells from the TPD was followed by transient neutropenia and bone marrow hypocellularity that gave way to a complete remission with recovery of full UCB chimerism.

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Viral specific CTL

Viral infections post-HSCT are an increasingly recognized cause of post-transplant morbidity and mortality. Adoptive immunotherapy to reconstitute antiviral immunity with in vitro expanded virus-specific CTLs is an attractive option for prophylaxis and/or treatment (Leen & Heslop, 2008). Infusion of these products should be safe, because the expansion protocol needed to selectively increase the small numbers of virus-specific T cells present in the PB mononuclear cells should also serve to deplete alloreactive T cells. This approach has been used to prevent and treat EBV-related complications of HSCT (Gustafsson et al, 2000; Comoli et al, 2006, 2007; O’Reilly et al, 2007), as well as to prevent and treat post-transplant CMV reactivations and infections (Walter et al, 1995; Einsele et al, 2002a,b; Peggs et al, 2003; Micklethwaite et al, 2007). These are quite common after UCBT, mainly in adults and patients who develop GVHD, and refractoriness to anti-virals poses a serious problem. Different investigator groups are pursuing research to simplify the process of obtaining products suitable for treatment of CMV reactivations and infections, in order to make them broadly available for clinical use. New protocols based on relatively rapid selection of tetramer (Cobbold et al, 2005) and antigen-stimulated interferon (IFN)-secreting T cells (Feuchtinger et al, 2004, 2005, 2006) may facilitate the adoptive transfer of donor derived CMV-specific T cells. Preliminary studies have shown that virus-specific T cells isolated by the two methods are safe and effective when transferred to patients with active infection. The main inconvenience of the two methods is that both require large volumes of blood (lymphopheresis products), although regular blood donation could suffice for selection of antigen-stimulated IFN-secreting T cells (Rauser et al, 2004).

Effective treatment of refractory CMV disease was recently described in a recipient of a ‘dual transplant’ following the infusion of CMV specific CTL from other donors (Schöttker et al, 2008). One of our ‘dual transplant’ patients, who was CMV seropositive prior to the transplant and whose TPD was his CMV seronegative haploidentical son, developed refractory CMV disease. Following the infusion of lymphocytes enriched for CMV-specific CTL (obtained by the procedure of selection of antigen-stimulated IFN-secreting T cells) from a different donor, the patient had a flare-up of cutaneous GVHD, due to the cells of the CB graft, that was controlled with immunosuppressive medication and infusions of MSC from his son. Despite this he achieved resolution of the CMV disease and antigenaemia (M.N. Fernández, M. Rico, T. Martín-Donaire, R. Gonzalo-Daganzo, I. Sanjuán, C. Regidor, R. Cabrera, unpublished data) (Fig 8). We believe that these two observations support the use of adoptive immunotherapy with selected cells obtained from immunized voluntary donors to combat infectious complications in recipients of ‘dual transplants’. Further evaluation by means of properly designed clinical assays seems warranted. Of special interest for application to UCBT would be the production of CMV-specific T cells from the naive UCB T cells (Savoldo et al, 2002; Comoli et al, 2006; Park et al, 2006).

image

Figure 8.  Response of CMV infection in a recipient of a ‘dual transplant’ to infusions of CMV-sp lymphocytes from another donor. A 45-year-old male AML patient, who was CMV-seropositive, received a CBT with co-infusion of MHSC and MSC from his 20-year-oldCMV-seronegative son. ANC recovered on day +11, but the CBU failed to engraft and he received a second CB unit on day +32 after a course of fludarabine and Timoglobulina. This unit engrafted and full chimerism was achieved on day +53. CsA was then discontinued. Within this interval he had cutaneous grade I aGVHD that responded to topical steroids, haemorrhagic cystitis due to polyoma virus and persistent CMV antigenemia despite treatment with antivirals. He was able to leave the hospital on day +64 but he was readmitted 1 month later with severe diarrhoea due to CMV infection, without evidence of aGVHD. As he only had a partial response to high doses of i.v. Gancyclovir, CMV-sp lymphocytes from an unrelated HLA mismatched donor were infused. Cutaneous grade II aGVHD began 5 d after the infusion, and required treatment with immunosuppressor drugs (steroids, CsA MM) and three infusions of MSC from his son. Symptoms of the CMV infection and antigenemia disappeared but because of persistent manifestations of aGVHD he had to be continued under immunosuppressive medication at hospital dismissal. The patient remained in full CB chimerism and biopsies from the GVHD skin lesions showed presence or DNA from the second CB unit, no DNA from the donor of MSC and CMV-sp lymphocytes being detected. He died 1 month later in his local hospital because of aetiologically undiagnosed interstitial pneumonitis. The graphs show the evolution of CMV antigenemia and number of circulating T CMV-sp T cells, identified as derived from the donor of the CMVsp lymphocytes (the patient was in full CB chimerims both in BM and PB granulocytes and mononuclear cells).

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Tumour specific T-cells

The GVT effect observed after allogeneic HSCT and donor lymphocyte infusions strongly suggests that T lymphocytes play a major role in the rejection of leukemic cells. T-cell therapy after allogeneic HSCT requires the separation of the GVT and GVHD effects to allow the reconstitution of adoptive immunity against infectious agents. Tumour antigen-targeted immunotherapy may be an efficient procedure to eliminate residual tumour stem cells that may persist as a reservoir of primitive and chemo-resistant tumour cells.

Requirements for T-cell immunotherapy to be clinically useful include the identification and characterization of appropriate antigenic structures expressed by tumour stem cells. Antigens that have been identified as possible targets of T cell adoptive immunotherapy include viral antigens expressed by the EBV-associated malignancies, minor histocompatibility antigens (mHA), or leukaemia-associated antigens such as different epitopes of the Wilms’ tumour gene 1 (WT1), the proteinase-3 derived epitope peptide (PR1), or epitopes of fusion-proteins produced from oncogenic chromosomal translocations. EBV-associated post-transplant LPD is a highly immunogenic tumour that may prove amenable to control by TPD virus-specific CTL. Procedures for the in vitro selection of donor T-cell with high specificity for those markers or for their manipulation to enhance their specificity and efficacy, such as transfer of antigen-specific T cell receptors, are also required. Cell products with a sufficient number of highly enriched tumour-specific T cells are likely to be devoid of significant numbers of allo-reactive T cells carrying the risk of GVHD, but products of this quality are difficult to produce. The incorporation of suicide genes that allow the selective destruction of allo-depleted or antigen-selected cells after infusion is another approach to increase the safety and potential applicability of these therapies to patients (Robin et al, 2005; Gottschalk et al, 2006; Greiner et al, 2006; Riddell et al, 2006; Kennedy-Nasser & Brenner, 2007; Heemskerk et al, 2008; Merlo et al, 2008). It is conceivable that these kinds of T-cell immunotherapy products derived from voluntary donors could prove useful for prophylactic or therapeutic use in the setting of UCBT.

T-regs

The possibility of using donor T regs as a tool to modulate immune reconstitution to a favourable balance between development of adaptive immunity, the preservation or enhancement of GVT effect and minimization of GVHD risk, is presently under investigation by different groups (Hoffmann et al, 2004; Rezvani et al, 2006; Figueroa-Tentori et al, 2008). The possible usefulness of these cells in UCBT recipients warrants investigation.

Future perspectives

  1. Top of page
  2. Summary
  3. 2009 general perspective of unrelated UCBT as a modality of HSCT: pros and cons and the risk of engraftment failure
  4. Clinical study of ‘Dual UCB/TPD–MHSC Transplants’: a strategy to increase likelihood of UCBT engraftment with single units of relatively low cell content and HLA mismatched
  5. How do ‘dual transplants’ compare to double UCB transplants?
  6. Use of other TPD cells in patients recipients of ‘dual transplants’
  7. Future perspectives
  8. Acknowledgements
  9. Funding
  10. References

UCBT has evolved into an effective strategy for the treatment of a number of haematological malignancies and non-malignant disorders. Achievement of high rates of engraftment is a pre-requisite for favourable long-term outcomes. Although further research may result in improved rates of engraftment, full UCB chimerism and less toxicity related to the conditioning regimens, the strategy of ‘dual transplants’ is an approach that results in early recovery of circulating granulocytes and high rates of UCBT engraftment and full chimerism. In this regard, it is a useful tool for making UCBT with single units of relatively low cell content feasible in adults, who represent the greatest demand for HSC transplants.

Nevertheless, recipients of UCBT have long-lasting susceptibility to a wide array of serious and often lethal infections, because of late reconstitution of adaptive immunity. Many of these are not amenable to available chemical antimicrobials either because of poor intrinsic therapeutic efficiency or because of deficient development of the immune reaction required to consolidate their antimicrobial effects. Thus, at this time, enhancement of the post-transplant immune reconstitution, mainly of the T cell component, is most urgently needed to improve long-term outcome of UCBT. Faster and better recovery of adaptive cellular immunity, without increasing risk of GVHD, is required to improve the long term efficiency of UCBT as a treatment for malignancies and other diseases. Moreover, for the treatment of neoplastic diseases, concomitant preservation or enhancement of the GVT effect, closely linked to the NK and T cell subpopulations, is also needed. The ‘dual transplant’ procedure could allow sparing a fraction of the CB unit for later infusion, once full CB chimerism has been achieved. This approach could allow to investigate the effects of late infused lymphocytes on the different immune reconstitution related manifestations.

As is also the case for other modalities of HSCT, UCBT are clearly changing engineered cell therapy procedures.

Acknowledgements

  1. Top of page
  2. Summary
  3. 2009 general perspective of unrelated UCBT as a modality of HSCT: pros and cons and the risk of engraftment failure
  4. Clinical study of ‘Dual UCB/TPD–MHSC Transplants’: a strategy to increase likelihood of UCBT engraftment with single units of relatively low cell content and HLA mismatched
  5. How do ‘dual transplants’ compare to double UCB transplants?
  6. Use of other TPD cells in patients recipients of ‘dual transplants’
  7. Future perspectives
  8. Acknowledgements
  9. Funding
  10. References

Members of the Servicio de Hematología, Hospital Universitario Puerta de Hierro:

Clinical Team: R. Cabrera, C. Regidor, I. Sanjuán, R. Forés, J. A. García-Marco, G. Bautista, E. Ruiz, B. Navarro, S. Gil, E. Ojeda, I. Krsnik, J. Gayoso, A. de la Iglesia, I. Vicuña and A. Sebrango.

Research Laboratory Team: E. Magro, R. Gonzalo-Daganzo, T. Martin-Donaire, M. Rico, N. Panadero, Y. Gutiérrez, M. García-Berciano, R. Sánchez, N. Pérez-Sanz and N. Polo.

Nursing staff.

Other contributors: I. Millán (Statistician, HUPH, Madrid), A. Madrigal, S. Querol, A. M. Little and A. McWhinnie (ANRI, London), J.A. Bueren and M. Ramírez (CIEMAT, Madrid). G. Delclos (Houston, Tx, USA) is acknowledged for his critical review of the manuscript.

References

  1. Top of page
  2. Summary
  3. 2009 general perspective of unrelated UCBT as a modality of HSCT: pros and cons and the risk of engraftment failure
  4. Clinical study of ‘Dual UCB/TPD–MHSC Transplants’: a strategy to increase likelihood of UCBT engraftment with single units of relatively low cell content and HLA mismatched
  5. How do ‘dual transplants’ compare to double UCB transplants?
  6. Use of other TPD cells in patients recipients of ‘dual transplants’
  7. Future perspectives
  8. Acknowledgements
  9. Funding
  10. References
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