**5. Preparation of patient**

The clinical condition of patients undergoing stem cell transplantation varies enormously depending on diagnosis, age, previous treatment, and organ damage. Once the decision to transplant has been made and a donor selected each organ system should be assessed so that any organ damage is known about prior to transplant and therefore potential harmful effects of chemotherapy, risks for GVHD and recurrence of infections can be anticipated. A full medical history should be taken and a physical examination performed. It is useful to perform the assessment at least 6 weeks before the transplant date in order that any necessary investigations can be arranged. A checklist is essential (Figure 2). It is helpful to perform microbiology screening for resistant bacteria e.g. methicillin resistant staphylococcus aureus (MRSA) and glycopeptide resistant enterococci (GRE/VRE). This will depend on unit policy.


on the mother:

Anti-HIV-1,2 HBsAg

Anti HBc (hepatitis B core antibody) Anti-HCV-Ab (anti-hepatitis C antibody)

originate from those areas.

**5. Preparation of patient** 

testing is required:

with Treponema pallidum (Anti-T. pallidum)

Nucleic acid testing for HIV (HIV-NAT) Nucleic acid testing for hepatitis B (HBV-NAT) Nucleic acid testing for hepatitis C (HCV-NAT)

the donor's parents originate from those areas.

below:

For cord blood donations at the time of cord collection tests for the following are performed

The current minimum serology testing requirements for these products are summarised

HTLV-I antibody testing for maternal donors living in, or originating from, high-incidence areas or with sexual partners originating from those areas, or where the donor's parents

Furthermore, if the cord blood unit has been stored for more than 180 days the additional

Nucleic acid testing for HTLV-I (HTLV-I-NAT) of maternal donors living in, or originating from, high-incidence areas or with sexual partners originating from those areas, or where

Confirmatory tests 3 to 6 months after delivery are also performed. The health of the baby is also assessed. Cord blood banks will supply the transplant centre with the required viral status of the mother and sometimes the cord blood itself. The cell doses contained within the cord donation and cell viability are also recorded. They will also perform extra tests for confirmatory tissue typing and virology and serology on small aliquots of the cord. Some centres may wish to perform their own confirmatory typing. A small sample is usually

The clinical condition of patients undergoing stem cell transplantation varies enormously depending on diagnosis, age, previous treatment, and organ damage. Once the decision to transplant has been made and a donor selected each organ system should be assessed so that any organ damage is known about prior to transplant and therefore potential harmful effects of chemotherapy, risks for GVHD and recurrence of infections can be anticipated. A full medical history should be taken and a physical examination performed. It is useful to perform the assessment at least 6 weeks before the transplant date in order that any necessary investigations can be arranged. A checklist is essential (Figure 2). It is helpful to perform microbiology screening for resistant bacteria e.g. methicillin resistant staphylococcus aureus (MRSA) and glycopeptide resistant enterococci (GRE/VRE). This will depend on unit policy.

taken at the time of thawing for DNA analysis for post transplant chimerism studies.

A validated testing algorithm to exclude the presence of syphilis/active infection

With permission from Kath Grey. Liaison team nursing sister. Great North Children's Hospital, Newcastle upon Tyne

**Figure 2.** Checklist for patients requiring an immunology assessment for HSCT

A number of infectious agents, particularly viruses, can now be detected by sensitive molecular techniques such as polymerase chain reaction (PCR) at an early stage of the infectious process. This in turn means that pre-emptive therapy can be given before organ damage such as life threatening pneumonitis or hepatitis, occurs. Such early detection together with treatments such as cidofovir for Adenovirus has led to a dramatic improvement in outcome following viral infection.

Many children with PID fail to thrive and are malnourished and it is important to maximise nutritional status prior to transplant, which may mean high calorie enteral feeding via nasogastric tube or parenteral nutrition through a central venous catheter.

Stem Cell Transplantation for Primary Immunodeficiency 13

area under the curve: six of them had AUCs below the target range, highlighting the difficulty in giving busulphan to this young group of children despite careful therapeutic

Reduced-intensity conditioning regimens using drugs such as fludarabine and melphalan have diminished treatment-related toxicity in some PID patients,[3] but toxicity remains a problem for children under one year of age,[34] and cardiac toxicity is associated with melphalan[35]. Minimal-intensity conditioning with fludarabine and low dose cyclophosphamide can reduce toxicity even further, but may be associated with poor donor myeloid chimerism or an increased incidence of GVHD.[36] Consequently, new conditioning regimens for PIDs have been developed that give adequate myeloablation but less toxicity, particularly in patients under one year of age. Treosulfan (l-treitol-1, 4-bismethanesulfonate) is the pro-drug of l-epoxybutane, a water-soluble bifunctional alkylating agent with myeloablative and immunosuppressive properties,[37] and is effective in HSCT conditioning with less toxicity, particularly in VOD, compared to busulphan. A recent study of 70 children in the UK, transplanted for various PIDs and given treosulfan with either fludarabine or cyclophosphamide, showed an overall survival of 81%. Of 46 children less than 12 months of age at HSCT, only eight died (overall survival 83%). Two children had VOD when treosulfan was given in combination with cyclophosphamide, but none when it was given with fludarabine. In the treosulfan cohort, there was no cardiactoxicity, such as seen with melphalan or pulmonary fibrosis as seen with busulphan[38-40]. T cell chimerism was significantly better when treosulfan was given in combination with fludarabine, and although numbers were small, there was a suggestion that chimerism was better when PBSCs were used as the stem source. Conversely, patients receiving cord blood stem cells in conjunction with serotherapy had a tendency to more mixed donor chimerism. While donor myeloid chimerism was sufficiently high to cure most patients with CGD and WAS with all donor types/stem cell sources, where 100% donor chimerism might be preferred in all cell lineages—for example, WAS[20] — then the use of PBSCs or full myeloablation with busulphan might be required. Early results using PBSCs with treosulfan and fludarabine look promising in terms of good chimerism without an increased incidence of greater than

**Figure 3.** Chemotherapy regimens, increasing in intensity from left to right

monitoring.[33]

Some diseases such as IPEX syndrome require immunosuppression to control autoimmune enteropathy and it is important to suppress any active inflammation prior to transplant, but also to minimise immunosuppression in order to reduce risk of infection.

Familial haemophagocytic lymphohistiocytosis is a genetically determined disorder characterized by the early onset of fever, hepatosplenomegaly, central nervous system disease, thrombocytopenia, coagulation disorders, and haemophagocytosis. It is caused by genetic defects that impair T cell–mediated and natural cytotoxicity. Chemotherapy and/or immunotherapy-based treatments can achieve remission, but HSCT is the only curative option. Outcome of HSCT is much more favourable if active disease is controlled prior to transplantation[28].

Sometimes a combination of antimicrobials and immunosuppression may be the optimal treatment. For example, Leiding et al have described 9 cases of severe life threatening liver abscesses in patients with CGD that progressed despite appropriate antibacterial therapy and drainage but responded to moderately high doses of corticosteroids tapered over several months.[29]

It is essential to address family and psychosocial issues and so each patient should be referred to a social worker and psychologist. Each unit will have their own system for information giving including written information about the transplant procedure, a visit to the unit and a home visit. It may be appropriate for patients to meet the dietician and physiotherapist prior to transplant. Fertility issues need to be discussed. In particular sperm banking should be arranged if appropriate. It is vital that written consent is taken once the patient/family are fully aware of all the aspects of the transplant.

#### **6. Conditioning**

Conditioning aims to create space in the recipient marrow niche to enable donor stem cells to engraft more easily. A range of conditioning regimens are available from non conditioning, through immunosuppressive regimens to myeloablative combinations (Figure 3).

For many years, myeloablative chemotherapy with busulphan and cyclophosphamide was given prior to HSCT for PIDs. However, busulphan is associated with significant toxicity including veno-occlusive disease (VOD). A strong correlation between blood levels of cyclophosphamide metabolites and VOD has previously been shown due to depletion of glutathione from the liver[30]. Combinations of cyclophosphamide with reduced-dose busulphan may also lead to severe hepatic toxicity and VOD [31, 32]. The combination of fludarabine and full dose busulfan has gradually replaced the combination of busulfan and cyclophosphamide in most centres. Malar et al. have recently reported on the importance of therapeutic drug monitoring for intravenous busulphan therapy in 34 paediatric patients. Seven children all weighing less than 12 kg had VOD despite not exceeding the targeted area under the curve: six of them had AUCs below the target range, highlighting the difficulty in giving busulphan to this young group of children despite careful therapeutic monitoring.[33]

**Figure 3.** Chemotherapy regimens, increasing in intensity from left to right

12 Immunodeficiency

transplantation[28].

several months.[29]

**6. Conditioning** 

Many children with PID fail to thrive and are malnourished and it is important to maximise nutritional status prior to transplant, which may mean high calorie enteral feeding via

Some diseases such as IPEX syndrome require immunosuppression to control autoimmune enteropathy and it is important to suppress any active inflammation prior to transplant, but

Familial haemophagocytic lymphohistiocytosis is a genetically determined disorder characterized by the early onset of fever, hepatosplenomegaly, central nervous system disease, thrombocytopenia, coagulation disorders, and haemophagocytosis. It is caused by genetic defects that impair T cell–mediated and natural cytotoxicity. Chemotherapy and/or immunotherapy-based treatments can achieve remission, but HSCT is the only curative option. Outcome of HSCT is much more favourable if active disease is controlled prior to

Sometimes a combination of antimicrobials and immunosuppression may be the optimal treatment. For example, Leiding et al have described 9 cases of severe life threatening liver abscesses in patients with CGD that progressed despite appropriate antibacterial therapy and drainage but responded to moderately high doses of corticosteroids tapered over

It is essential to address family and psychosocial issues and so each patient should be referred to a social worker and psychologist. Each unit will have their own system for information giving including written information about the transplant procedure, a visit to the unit and a home visit. It may be appropriate for patients to meet the dietician and physiotherapist prior to transplant. Fertility issues need to be discussed. In particular sperm banking should be arranged if appropriate. It is vital that written consent is taken once the

Conditioning aims to create space in the recipient marrow niche to enable donor stem cells to engraft more easily. A range of conditioning regimens are available from non conditioning,

For many years, myeloablative chemotherapy with busulphan and cyclophosphamide was given prior to HSCT for PIDs. However, busulphan is associated with significant toxicity including veno-occlusive disease (VOD). A strong correlation between blood levels of cyclophosphamide metabolites and VOD has previously been shown due to depletion of glutathione from the liver[30]. Combinations of cyclophosphamide with reduced-dose busulphan may also lead to severe hepatic toxicity and VOD [31, 32]. The combination of fludarabine and full dose busulfan has gradually replaced the combination of busulfan and cyclophosphamide in most centres. Malar et al. have recently reported on the importance of therapeutic drug monitoring for intravenous busulphan therapy in 34 paediatric patients. Seven children all weighing less than 12 kg had VOD despite not exceeding the targeted

through immunosuppressive regimens to myeloablative combinations (Figure 3).

nasogastric tube or parenteral nutrition through a central venous catheter.

also to minimise immunosuppression in order to reduce risk of infection.

patient/family are fully aware of all the aspects of the transplant.

Reduced-intensity conditioning regimens using drugs such as fludarabine and melphalan have diminished treatment-related toxicity in some PID patients,[3] but toxicity remains a problem for children under one year of age,[34] and cardiac toxicity is associated with melphalan[35]. Minimal-intensity conditioning with fludarabine and low dose cyclophosphamide can reduce toxicity even further, but may be associated with poor donor myeloid chimerism or an increased incidence of GVHD.[36] Consequently, new conditioning regimens for PIDs have been developed that give adequate myeloablation but less toxicity, particularly in patients under one year of age. Treosulfan (l-treitol-1, 4-bismethanesulfonate) is the pro-drug of l-epoxybutane, a water-soluble bifunctional alkylating agent with myeloablative and immunosuppressive properties,[37] and is effective in HSCT conditioning with less toxicity, particularly in VOD, compared to busulphan. A recent study of 70 children in the UK, transplanted for various PIDs and given treosulfan with either fludarabine or cyclophosphamide, showed an overall survival of 81%. Of 46 children less than 12 months of age at HSCT, only eight died (overall survival 83%). Two children had VOD when treosulfan was given in combination with cyclophosphamide, but none when it was given with fludarabine. In the treosulfan cohort, there was no cardiactoxicity, such as seen with melphalan or pulmonary fibrosis as seen with busulphan[38-40]. T cell chimerism was significantly better when treosulfan was given in combination with fludarabine, and although numbers were small, there was a suggestion that chimerism was better when PBSCs were used as the stem source. Conversely, patients receiving cord blood stem cells in conjunction with serotherapy had a tendency to more mixed donor chimerism. While donor myeloid chimerism was sufficiently high to cure most patients with CGD and WAS with all donor types/stem cell sources, where 100% donor chimerism might be preferred in all cell lineages—for example, WAS[20] — then the use of PBSCs or full myeloablation with busulphan might be required. Early results using PBSCs with treosulfan and fludarabine look promising in terms of good chimerism without an increased incidence of greater than

grade II GVHD. Prospective studies are needed comparing treosulfan/ fludarabine with busulphan and fludarabine,[41-43] where early reports also indicate reduced transplantrelated toxicities and excellent survival. Long-term follow-up is needed to see if this "modified intensity" regimen results in less long-term toxicity and, in particular, infertility, as the gonadal toxicity of busulphan is already well documented[44]. Radioimmunotherapy, in which targeted irradiation of the bone marrow is achieved by using radiolabeled monoclonal antibodies, may be used as a potent myeloablative agent with low intrinsic organ toxicity. Schulz et al. recently reported on the use of radioimmunotherapy with 90Yanti-CD66 for conditioning in 30 pediatric patients undergoing HSCT, including 16 with non-malignant disorders with high co-morbidities. Patients received radioimmunotherapy with fludarabine or melphalan alone or in combination, usually with ATG; one patient was given radioimmunotherapy alone. A highly favorable ratio of marrow dose to other organ dose was demonstrated, and stable engraftment was achieved with complete donor chimerism in 13 out of 16 (81%) patients. One patient with Griscelli syndrome and who had secondary graft failure after the first HLA haploidentical transplantation achieved stable complete donor chimerism with radioimmunotherapy alone[45].

Stem Cell Transplantation for Primary Immunodeficiency 15

passing the HSC source through a magnetic column[48]. The purified CD34+ HSC fraction is infused into the patient. By using this method, 4 log depletions of T-lymphocyte numbers can be achieved. There are important differences between CD34+ stem cell–selected and T lymphocyte–depleted bone marrow. Although the residual T-lymphocyte count in anti-CD52–treated marrow can be relatively high, very few of the T lymphocytes remain viable because they are still coated with anti-CD52 when infused into the recipient and are then destroyed by complement-mediated lysis. In the anti-CD34 selected HSC product, although the number of T lymphocytes infused into the patient might be very low, those that are infused are viable and could cause GvHD. The anti-CD52– treated product contains component blood cell precursors and cells already in early differentiation from the stem cell, as well as other stromal factors that might aid engraftment of HSCs into the bone marrow

An alternative means of stem cell enrichment has been used with the aim of achieving an optimal balance between the competing risks of GVHD, poor engraftment and delayed immune reconstitution. By targeting T- and B- cells specifically CD3/CD19 depleted grafts not only retain CD34+ stem cells but also CD34 negative progenitors, natural killer,

Further developments are now focusing on the depletion of TcRαβ+ T cells which can prevent GvHD after allogeneic stem cell transplantation from HLA non identical donors and may lead to more rapid immune reconstitution than CD3+ depletion of grafts. In contrast to

A successful HSCT procedure in patients with primary immunodeficiency should result in a cure of the underlying defect with normal immuno-reconstitution leading to normal immune function and a normal life. The likelihood of a successful outcome depends on the underlying diagnosis, degree of HLA matching from the donor, and pre existing end organ damage. Overall the outcome for patients with SCID is better than that for patients with other PID and has improved over time[8]. The likelihood of cure following HSCT is up to 90% in those SCID patients with a genoidentical donor. Even for those with no HLA identical donor, survival following a T cell depleted haploidentical transplant is approaching 60%. Whilst infusion of the stem cell product without the use of chemotherapy eliminates the potentially fatal effects of conditioning including infection from aplasia and increased risk of pneumonitis and mucositis, the quality of engraftment is compromised. Thus for patients with common gamma chain or JAK3 SCID thymopoiesis may be achieved as T cell precursors engraft in the thymus[50], but B cell engraftment is unlikely and patients will remain on lifelong immunoglobulin replacement as recipient B cells are unable to produce IgM through the IL 21 mediated signalling path and do not undergo immunoglobulin class switching.[51] Patients thus remain at risk of bronchiectasis in the

space thus achieving better engraftment.

**8. Results** 

dendritic and graft-facilitating cells which enhance engraftment.

depletion of CD3+ T cells valuable TcRγδ+ T cells are spared.[49]

**8.1. Severe combined immunodeficiency** 

Antibodies to eliminate host stem cells prior to transplantation could mean that toxic conditioning regimens would not be needed. Administration of ACK2 and an antibody that blocks c-Kit function led to transient removal of >98% of endogenous HSCs in immunedeficient mice[46]. Subsequent transplantation with donor HSCs led to chimerism levels up to 90% compared to only 3% in those without this preconditioning. The pharmacological agent AMD3100, which is a CXCR4 inhibitor, has been shown to induce egress of HSCs out of the marrow and improve levels of donor HSC engraftment relative to untreated recipients[47]. Plerixafor, a CXCR4 inhibitor, may also be a more effective mobilizer of stem cells for donors of PBSCs than granulocyte colony stimulating factor (G-CSF), allowing larger doses of stem cells to be administered, which may lead to more rapid engraftment.

#### **7. Methods of T cell depletion**

In 1981 the introduction of T lymphocyte depletion to remove of alloreactive lymphocytes from the bone marrow source enabled transplantation across HLA barriers. This meant that patients without an HLA-identical donor could receive T lymphocyte depleted marrow from an HLA-haploidentical parent.

Profound T-cell depletion is a fundamental prerequisite for haploidentical donor transplantation to avoid severe GVHD and B cell depletion lessens the risk of Epstein Barr Virus (EBV)-related lymphoproliferative disease. Various methods have been used to remove viable T lymphocytes from the graft, including E-rosette lectin depletion and in vitro anti CD52 (CAMPATH-1M anti-lymphocyte antibody which is no longer available). Since the late 1990s European centers performing T lymphocyte–depleted HSCT for patients with PID have used CD34+ stem cell selection rather than T-lymphocyte depletion. The most commonly used method, the Miltenyi Clini-MACS system, uses an organic iron bead attached to an anti-CD34 antibody to isolate purified CD34+ HSCs from the other cells by passing the HSC source through a magnetic column[48]. The purified CD34+ HSC fraction is infused into the patient. By using this method, 4 log depletions of T-lymphocyte numbers can be achieved. There are important differences between CD34+ stem cell–selected and T lymphocyte–depleted bone marrow. Although the residual T-lymphocyte count in anti-CD52–treated marrow can be relatively high, very few of the T lymphocytes remain viable because they are still coated with anti-CD52 when infused into the recipient and are then destroyed by complement-mediated lysis. In the anti-CD34 selected HSC product, although the number of T lymphocytes infused into the patient might be very low, those that are infused are viable and could cause GvHD. The anti-CD52– treated product contains component blood cell precursors and cells already in early differentiation from the stem cell, as well as other stromal factors that might aid engraftment of HSCs into the bone marrow space thus achieving better engraftment.

An alternative means of stem cell enrichment has been used with the aim of achieving an optimal balance between the competing risks of GVHD, poor engraftment and delayed immune reconstitution. By targeting T- and B- cells specifically CD3/CD19 depleted grafts not only retain CD34+ stem cells but also CD34 negative progenitors, natural killer, dendritic and graft-facilitating cells which enhance engraftment.

Further developments are now focusing on the depletion of TcRαβ+ T cells which can prevent GvHD after allogeneic stem cell transplantation from HLA non identical donors and may lead to more rapid immune reconstitution than CD3+ depletion of grafts. In contrast to depletion of CD3+ T cells valuable TcRγδ+ T cells are spared.[49]
