Hematopoietic stem cell transplantation and cellular therapy

Histocompatibility testing

The MHC genes are mapped within a region called HLA located on the short arm of chromosome 6. HLA antigens are responsible for the rejection of foreign objects from the body. There are at least six major HLA loci tested as needed to identify best donors for allogeneic HSCT—A, B, C, DR, DQ, and DP. These loci are divided into two groups: class I antigens, HLA-A, HLA-B, and HLA-C, and class II antigens, HLA-DR, HLA-DQ, and HLA-DP. They are segregated by haplotype from an individual father and mother. The class I molecules are composed of an α chain and β2 microglobulin, are highly polymorphic, and are expressed on most nucleated cells and on platelets. The HLA class II loci are involved in exogenous antigen processing. The HLA class II antigens are heterodimeric cell surface molecules formed by the α and β chains, each of which is polymorphic. HLA terminology is designated by the World Health Organization nomenclature committee for factors of the HLA system and is updated at regular intervals ( https://www.ebi.ac.uk/ipd/imgt/hla/ ).

To assess the degree of HLA compatibility, donor and recipient need to be HLA “typed.” Typing methods have included serological (antigen) or molecular (allele) (with low, intermediate, or high resolution) approaches. The broadest designation of HLA type is based on serologic typing, with the highest specificity based on the actual DNA sequence.

Serological tests are rarely used. The following techniques have been used for the identification of class I and II types:

• Sequence-specific oligonucleotide probe

• Sequence-specific primer

• DNA sequencing [usually reserved to resolve any ambiguity that is not resolved by the previous methods. Routine next-generation sequencing (NGS)-based HLA typing has recently become more commonly used.]

HLA matching for the purpose of HSCT is generally confined to major class I and II loci, although it is increasingly appreciated that minor histocompatibility antigens also play important roles in the outcome of allogeneic HSCT. Any disparity at major HLA loci between the donor and the recipient requires vigorous immunologic intervention to avoid rejection, or, should engraftment occur, subsequent GVHD.

Donor selection

Any HLA-identical sibling, who does not have an anticipated risk for the underlying condition for which their sibling is undergoing transplant, should be considered a potential donor. ABO mismatch is not a contraindication, but if multiple donors are available, a donor with the same ABO type is preferred [major ABO mismatch (A or B or O) requires the removal of the red cells and/or plasma from the bone marrow graft prior to infusion]. In past studies, best outcomes have been associated with a fully matched HLA-matched sibling donor; however, only 20–25% of patients will have matched sibling donors within their family. When sibling donors are not available, volunteer unrelated donors who are HLA, A, B, C, and DRB1 compatible or minimally mismatched with the recipients are sought through a search of the international donor registries, including the National Marrow Donor Program.

An acceptable adult unrelated donor donating bone marrow or peripheral blood stem cells (PBSCs) should be matched via high-resolution DNA typing at least seven of eight alleles (HLA-A, B, C, and DRB1). When available, a higher degree of match (i.e., 10 of 10 alleles, including DQ, or 12 of 12, including DP, and matching as much as possible for HLADRB3/4/5 alleles) is desired.

Other alternatives include umbilical cord blood (UCB) donor, which has been matched via low-resolution typing of A and B with high-resolution DRB1 typing, at least four of six antigens (HLA-A, B, and DRB1), but better outcomes have been associated with matching at least six of eight antigens at the high-resolution typing level.

Bone marrow

Bone marrow harvesting is carried out using general or epidural anesthesia under sterile conditions.

The recommended cell dose and volume of marrow to be collected are determined by the recipient’s body weight and diagnosis, the type of graft manipulation (if any) that will occur, and the size of the donor.

The iliac crests (most common posterior and rarely anterior if an adequate number of cells are not obtained from posterior iliac crests) of the donor are prepared and draped. Approximately 5 mL of bone marrow is aspirated from each bone puncture until the required amount is obtained, with multiple bone punctures performed through a single skin puncture. The marrow is collected in a heparinized collection system. The minimum concentration of heparin (preservative-free) is 3–5 units/mL of bone marrow. The quantity of nucleated bone marrow cells required to ensure engraftment is 2–5×10 ⁸ cells/kg of recipient body weight with a goal of >4–5×10 ⁶ CD34+ cells/kg. The usual volume of marrow required to achieve this cell yield is 10–15 mL/kg of recipient body weight. The maximum amount one can take from a donor to avoid needing blood transfusion is 20 mL/kg donor weight. Marrow from children, especially infants, has a higher proportion of marrow-repopulating cells than marrow from older donors. The marrow is filtered through a filtering apparatus to remove bone and tissue fragments and placed in a blood transfer pack. In allogeneic transplants the bone marrow is then given to the recipient as an intravenous (IV) infusion over a period of a few hours.

Peripheral blood stem cells

PBSCs are the most commonly used source for an autologous graft, and bone marrow is reserved for cases where PBSC collection or mobilization is not feasible. PBSCs are collected from donors after stimulation with G-CSF (10 µg/kg per day) and have been successfully used to reconstitute hematopoiesis in recipients of autologous, syngeneic, and allogeneic grafts. Patients who receive >5×10 ⁶ CD34+ cells/kg engraft satisfactorily (minimum 2×10 ⁶ CD34+/kg required). In autologous donors a combination of myelosuppressive chemotherapy and G-CSF administration is more effective at mobilizing CD34+ cells. Plerixafor alone or in combination with G-CSF may be used in certain conditions for mobilization.

Plerixafor is usually used in patients or donors requiring PBSC collection who fail to mobilize, as defined by peripheral blood CD34+ cell counts <20 cells/µL following G-CSF-based mobilization protocols. Plerixafor used in children is off-label [not Food and Drug Administration (FDA) approved for use in children]. The standard dose is 0.24 mg/kg (maximum dose 40 mg) once daily subcutaneous for up to 4 consecutive days. Each dose should be given around 10 hours prior to apheresis. The use of high-dose Plerixafor 0.48 mg/kg once daily, to improve mobilization, has also been reported.

Umbilical cord blood

UCB has been collected, cryopreserved, and is used as the source of pluripotential HSCs when a related or unrelated stem cell donor is not available. UCB cells have increased proliferative capacity and decreased alloreactivity, with a lower incidence of GVHD, in addition to immediate availability. These properties, coupled with an absence of donor risk, is an advantage in the use of this source of HSC, but disadvantages include the inability to fully evaluate the genetic history of these donors, limited cell dose, and lack of feasibility of donor lymphocytes infusion if needed. For a single UCB transplant, the standard minimum cell dose must be equal to or >2.5×10 ⁷ TNC/kg of recipient weight. If the transplant is performed with two cord blood units, the preferred dose of the combined units is ≥3.0×10 ⁷ TNC/kg of recipient weight. A higher cell dose is generally preferred and especially when there is a greater degree of HLA mismatch between the cord unit(s) and the intended recipient. For cord blood the interaction between the cell dose available and HLA matching affects the selection strategy as follows:

• 1.

  Single unit:

  • a.

    The UCB unit is a 6/6 match with the recipient with a cell dose ≥2.5×10 ⁷ TNC/kg.

  • b.

    The UCB unit is a 5/6 matched with a cell dose ≥4.0×10 ⁷ TNC/kg.

  • c.

    The UCB unit is a 4/6 matched with a cell dose ≥5.0×10 ⁷ TNC/kg.

• 2.

  Double units should be adequately matched with each other and the recipient as discussed earlier.

ABO incompatibility

ABO incompatibility between donor and recipient is encountered in 25–30% of allogeneic transplants. A major incompatibility occurs when the recipient plasma contains isohemagglutinins directed against the donor RBC antigens (e.g., group O recipient, group A donor), and minor incompatibility occurs when the donor plasma contains isohemagglutinins directed against recipient RBC antigens (e.g., group A recipient, group O donor). Red cell depletion is required for major mismatches in order to minimize the amount of incompatible blood in the graft. After the transplant procedure, patients can be followed with immunohematologic testing for the appearance of donor-derived RBCs and changes in recipient isohemagglutinin titers.

Graft-versus-host disease

Activation of allogeneic T cells against recipient antigens is termed GVHD. All forms of allogeneic HSCT require approaches to reduce the risk of GVHD, most commonly in the form of GVHD prophylaxis using medications such as cyclosporine or tacrolimus. Reduction of T lymphocytes from allogeneic HSC postcollection decreases the risk of GVHD and is sometimes part of the approach to GVHD prevention. This can be achieved through various techniques, including monoclonal antibodies accompanied by complement-mediated lysis, immunotoxins, or immunomagnetic beads (complete or partial in vivo T-cell depletion may include administration of ATG or alemtuzumab). An alternative method is to administer post-HSCT cyclophosphamide to accomplish in vivo T-cell depletion following infusion of an unmanipulated graft. This approach is commonly used for HLA-haploidentical donor grafts but is increasingly being used for other graft types as well.

Purging of malignant cells

Purging can be performed to remove malignant cells. This can be achieved through the use of monoclonal antibodies combined with complement, monoclonal antibodies linked to toxins or various chemotherapy drugs. This has been used in studies of autologous HSCT for neuroblastoma, and published data from a phase 3 randomized trial showed that purging of PBSC did not improve outcome. Because of this and other studies, purging tumor from autologous PBSC is not commonly practiced.

Commonly used conditioning regimens