Introduction

Interest in BMT from haploidentical donors arises from the immediate availability of a one haplotype-mismatched donor for virtually all patients, particularly those who urgently need transplantation. Most adults with ALL or AML relapse especially when they have unfavourable cytogenetics at diagnosis, when they do not achieve CR after the first induction cycle and when they are in second or later remission.^{1, 2, 3, 4} Under these circumstances, an allogeneic haematopoietic stem cell transplantation (HSCT) is preferred as post-remission therapy but the ideal donor, an HLA-identical sibling, is available for 30% of patients.

The only option is transplantation from an alternative donor. In the international registries the probability of finding a matched unrelated donor (MUD) ranges from 10% in poorly represented ethnic groups to 60% in Caucasians.⁵ Many patients relapse while the HLA typing is in progress or while waiting to start transplant procedures. Unrelated cord blood (UCB) offers the advantages of easy procurement and immediate availability.^{6, 7} For adults, the great divergency between body weight and the number of haematopoietic cells in a cord blood unit, particularly if associated with a two antigen mismatch, increases the risk of graft failure and delays haematopoietic reconstitution. Even though transplantation of two cord blood units is providing promising results,⁸ the only feasible option for many adults is transplantation from a haploidentical donor.

Besides immediate donor availability, mismatched family donors offer several other advantages: (1) ability to select the best of many relatives on the basis of age, infectious disease status and natural killer (NK) cell alloreactivity;⁹ (2) optimal graft composition and (3) immediate access to donor-derived cellular therapies. Furthermore, for nearly all patients who reject the graft, the haploidentical transplant offers the advantages of another family member as donor or a second graft from the original donor.

Basic and clinical research on haploidentical transplantation has been proceeding for over 20 years and has spread to centres worldwide. Some centres focused on manipulating the conditioning regimens and post transplant immune suppression; others, such as our centre in Perugia, Italy, concentrated on manipulating the graft. There is consensus that rejection and GVHD, which were inevitably linked to transplantation across the HLA barrier, have been resolved. Best survival rates were around 55% for AML and 28% for ALL in adults transplanted in CR; best rates in children were 47% for acute leukaemia and 87% for non-malignant diseases.¹⁰ Poor post transplant immune reconstitution and infection-related mortality remain open problems which are discussed in depth below.

Engraftment and GVHD

Despite proof that a haploidentical graft could be successfully transplanted without causing GVHD in SCID patients receiving a lectin-separated BMT from haploidentical parents,¹¹ unmanageable T-cell alloreactions across the HLA barrier caused a high incidence of severe GVHD and rejection of extensively T cell-depleted BMT in leukaemia patients.^{12, 13} Enhancing immunosuppression and myeloablation did not ensure engraftment.^{14, 15, 16, 17, 18, 19} Removing under two log T cells from donor marrow improved the engraftment rate but required post transplant immunosuppression to prevent GVHD. A series of clinical trials administered TBI-based myeloablative conditioning, a graft with partial ex vivo T-cell depletion by T10B9 monoclonal antibody in 143 patients or by OKT3 in 58 patients. GVHD prophylaxis also included antithymocyte globulin (ATG), cyclosporine and steroids. Engraftment was successful in 98% of patients; grades II–IV acute GVHD and chronic GVHD were 13 and 15%, respectively.¹⁴

In 1993, we were the first to produce a megadose of human haematopoietic stem cells by adding G-CSF-mobilized PBPCs to bone marrow cells,²⁰ using a lectin-based technique to ensure over three log T-cell depletion. Primary engraftment was achieved in 16/17 patients with end-stage leukaemia. Without post transplant immune-suppressive treatment, severe acute GVHD occurred in only one patient and there were no cases of chronic GVHD.

Animal models had shown that transplantation of high doses of T-depleted marrow cells engrafted without causing GVHD as they reduced the frequencies of anti-donor CTL precursors (p_s) in vivo, probably through deletion mediated by tumour necrosis factor-.^{21, 22, 23, 24} Cells within the CD34⁺ cell population exhibited 'veto' activity, that is, in bulk-mixed lymphocyte reactions they neutralized specific CTL-p_s directed against their antigens but not against a third party.^{22, 23}

In 1995, fludarabine was substituted for cyclophosphamide to minimize extra-haematological toxicity of the TBI-based conditioning regimen,^{25, 26} and CD34⁺ cells were positively selected from leukapheresis products to reduce T cells to one log less than in the previous series. The final inocula contained a median of 10 10⁶ per kg CD34⁺ cells and 2 10⁴ T cell per kg. The engraftment rate was 95% with no GVHD.²⁶ The threshold dose of 2 10⁴ CD3⁺ cells per kg prevented severe GVHD as long as it was associated with ATG in the conditioning because ATG has a plasma half-life of several days and could exert in vivo T-cell depletion.^{27, 28, 29}

In 1999, we started using the automated Clinimacs device (Miltenyi Biotech, Bergisch Gladbach, Germany) which, besides providing a good yield of CD34⁺ cells, ensured a median of 4.5 log T-cell depletion and 3.2 log B-cell depletion,³⁰ which helps prevent EBV-related lymphoproliferative disorders in recipients of an extensively T cell-depleted HSCT after a conditioning regimen with ATG.^{31, 32} This automated graft processing system, which provides reliable and reproducible yields, has contributed to make extensively T cell-depleted haploidentical transplantation feasible in any transplant centre.

In 255 adults with acute leukaemia treated under fludarabine-based protocols we achieved 95% primary engraftment rate, which rose to 98% when we included the few recipients of a second haplo-transplant. Severe acute and extensive chronic GVHD were largely prevented. At the San Raffaele Hospital, Milan (Italy), the same transplant protocol was associated with similar results in a cohort of patients with advanced stage haematological malignancies.³³

At the University of Tuebingen (Germany), positive selection of CD34⁺ cells and a myeloablative conditioning regimen were associated with high transplant-related mortality (TRM) in pilot studies.³⁴ Consequently, negative selection of G-CSF-mobilized PBPCs (CD3/CD19 depletion with anti-CD3- and -CD19-coated microbeads on a Clinimacs device) was used so as to infuse large numbers of CD34⁺ and CD34- cells, CD8⁺ T cells, NK cells and other accessory cells.³⁵ Their current HSCT protocol includes reduced intensity conditioning (RIC)—melphalan, thiotepa and fludarabine—followed by CD3/CD19-depleted PBSCs. OKT3 is used instead of ATG.^{36, 37} Mycophenolate mofetil was needed in patients who received more than 2.5 10⁴ CD3⁺ cells per kg. In 27 adults (median age 41 years) with high-risk leukaemia, they confirmed 100% full-donor engraftment. Patients (13/27; 48%) developed acute GVHD (nine grade II, two grade III and two grade IV).³⁷ Shifting from positive to negative PBPC selection re-introduces GVHD, which requires immunosuppressive therapy, thus counteracting the advantage of early post transplant immunological recovery and compromising the quality of life of long-term survivors. Furthermore, RIC does not guarantee leukaemia burden debulking and, as follow-ups are very short, no definitive conclusions can be drawn about relapse.

At the Dana Farber Cancer Institute, Boston (USA), co-stimulatory blockade was used to induce tolerance in haploidentical transplantation.³⁸ Engraftment was successful in 11/12 evaluable patients, 3 with acute GVHD. When results were updated to include another 12 patients,³⁹ graft failure occurred in 4.2%, aGVHD grade II–III in 35% and cGVHD in 4.2%. This approach appears to support engraftment but does not eliminate GVHD.

Other researchers focussed on haploidentical transplants without T-cell depletion.^{40, 41} At Peking University (China), Huang et al.⁴⁰ combined G-CSF-primed BM and unmanipulated PBSCs after myeloablative conditioning and post transplant immune suppression with mycophenolate and cyclosporine (CYA). All 171 patients (86/171 at high-risk) achieved full-donor chimaerism. At 100 days after transplantation, grades II–IV and III–IV acute GVHD developed in 55 and 23%, respectively. Acute GVHD grades II–IV occurred in 52/66 patients (78%) and grades III–IV in 9 (13%) who were three-loci mismatched with donors. Despite ATG in the conditioning and intense post transplant immunosuppression, the incidence of chronic GVHD was 73.6%, suggesting patients enjoy a poor quality of life and may be at risk of late TRM due to opportunistic infections and toxicity related to long-term immunosuppression.

At Durham University, NC (USA), Rizzieri et al.⁴² used unmanipulated PBSCs after a new RIC: alemtuzumab, fludarabine and cyclophosphamide (CY). Additional GVHD prophylaxis included mycophenolate and CYA. Of the 49 patients, 94% engrafted; 8% had secondary graft failure. Acute GVHD (grades III–IV) occurred in 8%. This immunosuppressive RIC was associated with a high engraftment rate and a relatively low incidence of GVHD. Long-term outcomes remain to be ascertained.

After successful canine experiments, high-dose post transplant CY was tested in a phase I trial to determine the minimal conditioning that ensured engraftment of an HLA-1 to -3 antigen-mismatched BMT.⁴³ A total of 13 patients with haematological malignancies were conditioned with low-dose TBI, fludarabineCY and received CY again on day +3 post transplant. Altogether 9 of 13 patients engrafted; 6 developed acute GVHD. Attempts to compensate low-intensity conditioning with post transplant immune suppression were once again associated with low engraftment rates and GVHD.

Several groups exploited the principle of tolerance induction resulting from in utero exposure to maternal antigens in haploidentical stem cell transplantation. Due to transplacental trafficking of maternal and foetal cells during pregnancy, maternal antigens induce mutual hypo-responsiveness and long-lasting feto-maternal microchimerism.⁴⁴ Non-T cell-depleted haploidentical HSCT from tolerized donors (typically mothers or non-inherited maternal antigen (NIMA) mismatched siblings) was successful but most patients developed acute and/or chronic GVHD.^{45, 46} In several Japanese transplant centres, myeloablative or non-myeloablative conditioning followed by unmanipulated transplants from microchimeric NIMA-mismatched donors resulted in a 56% incidence of acute GVHD grades II–IV in 34 evaluable patients. The risk of GVHD was significantly lower when the donor was NIMA mismatched in the GVHD direction.^{46, 47, 48} A large International Bone Marrow Transplant Registry (IBMTR) analysis showed that after unmanipulated haploidentical HSCT, the incidence of grades II–IV acute GVHD was related to haplotype inheritance; acute GVHD was significantly less frequent after transplants from NIMA-mismatched siblings, but TRM was significantly higher in transplants from a parent.⁴⁹

Leukaemia relapse

Extensive T-cell depletion might be expected to result in a weak or no GVHD–GVL effect which is conventionally achieved through T cell-mediated alloreactions directed against histocompatibility antigens displayed on recipient leukaemic cells.^{50, 51} Under the Perugia protocol, despite the lack of GVHD–GVL effect and unfavourable prognostic features at transplant, relapse rates were very low. Relapse occurred in respectively 18 and 30% of AML and ALL patients transplanted in any CR. In AML the cumulative incidence of relapse was significantly lower after transplantation from NK alloreactive donors (3 versus 47%; P<0.003).⁵² Relapse rates rose to 34 and 58%, respectively for AML and ALL patients who were in relapse at transplant and there was no difference whether the donor was NK alloreactive or not (32 versus 37%; P=NS).⁵²

Factors which may have contributed to control post transplant relapse include: a highly myeloablative conditioning regimen, which successfully debulked the leukaemia burden; no post transplant immunosuppression which may have allowed the few T cells in the graft to exert a sub-clinical GVHD–GVL effect; donor-versus-recipient NK cell alloreactivity which exerts a specific Graft-versus-AML effect in susceptible patients^{9, 52, 53, 54} and in T, but not B, phenotype ALL.⁵³ Engineering NK cells to express anti-CD19 chimaeric and activating receptors was reported to provide cytotoxicity against NK cell-resistant ALL.⁵⁵ Furthermore, Leung et al.⁵⁶ showed that ALL cells are susceptible to NK-cell lysis when donor NK cells expressed natural killer cell immunoglobulin-like receptor (KIRs) in the absence of cognate ligand in the recipient. In addition when NK-cell alloreactivity is combined with donor-activating KIR genetics, which protect against infection, a significant survival advantage is obtained.^{52, 54} Therefore, as donor-versus-recipient NK-cell alloreactivity contributes to prevent graft rejection, GVHD- and infection-related mortality such donors are preferred for all haploidentical transplants.

In the German series, 6/27 adults and 8/21 children relapsed after receiving negatively selected haploidentical grafts.³⁷ The Dana Farber approach was associated with a 12.5% relapse rate.³⁹ Unfortunately no definitive conclusions can be drawn because of the small patient cohorts and brief follow-ups. As far as regards unmanipulated haploidentical transplantation Peking University reported relapse rates ranging from 12% in standard-risk patients to 39% in high-risk patients.^{40, 41} In the Durham report, relapse was the main cause of death, occurring in 24/49 patients.⁴² Therefore, relapse rates, despite the incidence of GVHD, were the main reasons for transplant failure.

Other studies have suggested a T cell-mediated anti-leukaemic effect.^{57, 58, 59} A retrospective analysis on 201 patients with acute leukaemia who received partially mismatched HSCT from 1993 to 1999 at the South Carolina Cancer Center⁵⁹ showed 77 ALL and 76 AML survivors on day +59. Eighteen patients with high T-cell counts post transplant had better 5-year survival than those with normal or low counts (70.8 versus 19.6%; P=0.0001). Like macrophages and NK cells, T cells exhibit biological characteristics of innate immunity and recognize malignant cells through mechanisms that require no prior antigen exposure or priming. Since few reports have investigated T-cell recovery during post transplant immune reconstitution, it remains to establish whether these observations are related to graft processing procedures or donor-related factors.

TRM and post transplant immune re-building

After all forms of transplantation from alternative donors TRM, post transplant immune re-building and infectious mortality remain outstanding issues for several reasons: (1) conditioning-induced tissue damage prevents T-cell homing to peripheral lymphoid tissues, where T-cell memory is generated and maintained; (2) for months after transplant, immune recovery in adults with their decayed thymic function stems from expansion of mature T cells in the graft, and afterwards, from de novo production of naive T cells; (3) delays in immune construction are due to (1) GVHD and its immunosuppressive treatment in unmanipulated transplants and (2) T-cell depletion and ATG in the conditioning, which creates a deficit in T-cell immunity.^{60, 61, 62, 63} Administering G-CSF after T cell-depleted haploidentical transplant promotes Th-2 immune deviation which, unlike Th-1 responses, does not protect against fungi, bacteria and viruses.⁶⁴ Patients who did not receive G-CSF after transplant recovered CD4⁺ cells count faster and most post transplant CD4⁺ cell clones exhibited Th1-Th0 features.⁶⁴

The Perugia group reported 40% of patients died of non-relapse causes. Most deaths were caused by infections, mainly CMV and aspergillus.^{26, 30} TRM depended on disease stage at transplant and was significantly higher in patients transplanted in relapse (58 versus 36%; P=0.02). Due to the absence of chronic GVHD and its treatment the risk of life-threatening episodes plateaued after about 1 year, confirming that immune competence was completely recovered at this stage.

Under the Tuebingen protocol, 7/27 adult patients and 2/21 children died (infections in six; GVHD in one).³⁷ The two children died of non-specified causes, presumably transplant-related toxicity. The Dana Farber group reported early death in 50% of their 24 patients, the most common cause being bacterial or fungal infections.³⁹ Peking University reports a TRM of 9.1 and 12.7% at day 100, respectively in standard- and high-risk groups. TRM rises to 19.5 and 31.1% at 2 years.⁴⁰ Of the 39 non-relapse deaths 21 (54%) were due to opportunistic infections.

The Durham group achieved 10.2% TRM at 100 days after transplantation, projecting to 40% at 2 years.⁴² Causes include infections, lymphoma, haemolytic anaemia and neurotoxicity. Quantitative lymphocyte recovery through expansion of transplanted T cells and immunoscope analysis of the family of T cells were noted by 3–6 months only in patients who did not develop GVHD. Although RIC followed by unmanipulated PBSCs seems appealing because of the low TRM at 100 days post transplant, the 2-year probability of relapse mortality approaches 50% (24/49 patients).

Any further reduction in TRM after haploidentical transplantation will only be achieved by hastening post transplant immune recovery. However alloreactivity is attenuated, T cell-based adoptive therapy is problematic in the adult haploidentical transplant setting as infusion of grafts containing only a few more T cells than the threshold dose of 10⁴ per kg may cause lethal GVHD. Obstacles to the transfer of donor immunity are (1) the HLA barrier; (2) the need to transfer donor T cells early when patients are most vulnerable to GVHD; (3) lack of post transplantation GVHD prophylaxis because immune suppression would protect against the GVHD potential of infused T cells and (4) the increased susceptibility of adults to GVHD.

Despite this, several strategies are under investigation. The Perugia group has recently transferred donor pathogen-specific immune responses safely across the HLA barrier.⁶⁵ Donor T-cell clones raised against aspergillus fumigatus and CMV antigens were screened for cross-reactivity to host alloantigens by MLR. Nonhost-reactive clones, presumably devoid of GVHD potential, were pooled and infused into recipients soon after transplant. This study demonstrating the maximum tolerated dose of 1 million CD3⁺ cells per kg recipient body weight was clinically effective as CMV reactivation and aspergillus galactomannan antigenemia tended to disappear over time. In patients who were infused, the frequencies of anti-aspergillus and -CMV-specific T-cell clones increased rapidly unlike the control group, where they developed in vitro more than 9 months post transplant.⁶⁵ In other transplant centres similar results were achieved when ex vivo-expanded EBV-specific allogeneic CTL clones were used to prevent or manage EBV-associated diseases, including post transplant lymphoproliferative disorders.^{66, 67} The main drawbacks of both approaches are that they are time-consuming, labour intensive and, at present, unsuitable for routine clinical use as cloning and screening procedures do not always satisfy quality controls.

Genetic manipulation of donor lymphocytes with a suicide gene is a promising strategy that was developed in Milan.^{68, 69} Should GVHD develop, after genetic engineering of donor lymphocytes with the herpes simplex virus-thymidine kinase (TK) suicide gene, transduced cells can be eliminated by ganciclovir treatment. In 17 patients, TK cells provided protection against CMV reactivation and disease. Overall, the cumulative infectious mortality at 6 months post transplant was 12.5% with only 6% CMV-related mortality. This strategy may find a place in preventing relapse, selectively controlling GVHD and/or enhancing wide-spectrum immunological reconstitution after transplantation.⁷⁰

Results of ex vivo-expanded immunomodulatory cells (T-regs, NK/T-regs, MSCs and donor-derived NK),^{71, 72, 73, 74} adoptive transfer of allogeneic T cells that are specific for viral^{65, 67, 75, 76, 77, 78} or tumour antigens⁷⁹ appear promising. All these diverse approaches show that cell therapy is feasible after haploidentical transplantation and may help re-build immunity to infections.

Survival

Event-free survival (EFS) after haploidentical transplantation is closely related to disease and disease status at transplant and compares favourably with survival after MUD transplant in the same risk category.^{80, 81} In Perugia, EFS in 83 AML patients transplanted in CR, ranges from 35% for those in second or later CR to 50% for CR1, while 62 patients with ALL who were transplanted in CR have about 25% probability of surviving event-free. With no chronic GVHD, all these long-term survivors enjoy an excellent quality of life.

Patients in chemoresistant relapse had no option other than transplantation from a mismatched family donor. The 14% 10-year EFS for 64 end-stage AML patients is more than encouraging, but as only 5% of 46 ALL patients survive event free, we do not recommend haploidentical transplant in advanced stage ALL.

In a multivariate analysis, donor-versus-recipient NK-cell alloreactivity impacted favourably upon survival in AML patients.⁵² A total of 30 patients in any CR who received a transplant from an NK-cell alloreactive donor enjoy 67% EFS versus 18% in 31 patients transplanted from non-NK alloreactive donors (P=0.02). In 21 AML patients who were in relapse at transplantation, NK-cell alloreactivity was associated with better survival (30 versus 6%; P=0.04). In fact, we recommend transplantation for AML patients in relapse only if an NK alloreactive donor is available.

A preliminary report from the Tuebingen group that 14/27 patients (52%) survive at a median follow-up of 8 months³⁷ needs to be confirmed in a much larger series of patients with a longer follow-up.

Guinan et al. reported 5 of the original 12 patients were alive and in remission 4.5–29 months after transplantation which was updated to 8/24 long-term survivors (33.3%) at a median follow-up of 3.3 years.^{38, 39} All eight had an excellent performance score with good immune function; none was on chronic medication.

The Chinese study reported a 2-year probability of leukaemia-free survival as 68% for standard-risk patients and 42% for high-risk patients. When patients were stratified for age, survival was worse in patients over 35 (P=0.033), probably because of a higher incidence of GVHD-related mortality.^{40, 41}

In the Durham study, 75% of evaluable patients achieved CR after transplantation and 1-year survival was 31% (95% CI, 18–44%).⁴² Better results were obtained in a small group of 19 patients with standard risk that is remission or aplasia. Overall1-year survival was 63% (95% CI, 38–80%) with a 2.9-year median overall survival (95% CI, 6.2–48 months).

In the small series of 13 patients who received CY after transplantation, O'Donnell et al.⁴³ reported 6/13 patients were alive, 5 in CR at a median of 191 days post transplant.The most recent trials on haplotransplant are reported in Table 1.

Table 1 - T cell-depleted and unmanipulated haploidentical stem cell transplantation.

Full table

Conclusions

The past few years have seen growing interest in haploidentical transplantation, with several groups modifying conditioning regimens, graft processing and post transplant cellular immunotherapies in attempts to improve outcomes and provide convincing clinical evidence in favour of this transplant modality. The choice of the best alternative source of stem cells for individual patients without matched sibling donors is hampered by lack of randomized studies supplying data on outcomes after MUD, UCB and haploidentical transplants. Designing such a study is difficult because, at present, allocation of a patient to one of these three options could reflect a transplant centre's preferential use of a particular transplant modality rather than selection of the best donor for each individual patient.⁸² However, retrospective data analyses show survival rates overlap after haploidentical transplants for high-risk acute leukaemia and transplants from other alternative sources.^{80, 81, 82} It is worth noting that the probability of EFS after matched unrelated transplants is confounded by the gap between the number of activated searches and de facto transplants and many patients relapse and die while searching for an appropriate donor. Furthermore, independently of matching, many UCB transplants for adults were not performed because units did not contain the recommended threshold cell dose for engraftment.

Neither time lapse nor insufficient stem cell dose is an issue in the haploidentical transplant. Donors are found within the family for virtually all patients with no undue delay between decision-making and transplantation, which is a crucial factor in urgent cases. If the selected donor is a poor mobilizer, another mismatched family member is available as replacement. Although current data in most series refer to small, diverse patient populations, the main complications are GVHD in unmanipulated haploidentical SCT and delayed immune recovery in the T cell-depleted modality because rejection, GVHD and leukaemia relapse are no longer major issues in patients who are not transplanted in end-stage disease. In the absence of alloreactive T cells, the NK cell impacts beneficially upon outcomes. Donor-versus-recipient NK-cell alloreactivity strengthened the Gv-AML effect, improved engraftment and helped prevent GVHD independently of disease.^{52, 54}

As disease status at transplantation seems to be the main determinant of treatment failure, one is left with the question of whether transplant timing has become a determining factor in outcomes. It is to be hoped that transplant physicians will be less hesitant to treat patients in better condition and earlier disease stage, and that the mismatched transplant will be offered, not as a last resort, but as a routine option to high-risk acute leukaemia patients.

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Acknowledgements

I thank all the laboratory and technical staff, the attending physicians and nurses of the HSCT Unit at the University of Perugia, Italy, for their dedication and professional skills. Special thanks to Professor Massimo F Martelli for pioneering the mismatched transplant, Professor Yair Reisner for his expertise in transplant biology and Dr Geraldine Boyd for his help in writing this paper.