Stem cell transplantation during cancer.

Hematological malignancies account for approximately 9.5% of new cancers diagnosed annually. Lymphoma is the most frequent of all known categories of hematological malignancies. Worldwide, extensive research has focused on this type of cancer. However, new treatments are investigated in various clinical as well as pre-clinical studies. Hematopoietic stem cell transplantation (HSCT) is a recent and upcoming treatment strategy for patients with hematopoietic malignancies and inborn errors of metabolism or immune deficiencies. Recent studies have revealed that successful clinical outcome of this treatment strategy depends on multiple factors including the protocol applied, disease under treatment, health of the patient, source of the grafts, severity of complications such as graft versus host disease during grafting and associated infections. The scope of this review is to achieve greater understanding of various clinical effects of the disease and related mechanisms. The electronic database Pubmed was searched for pre-clinical as well as clinical controlled trials reporting efficacy of the HSCT against hematological malignancies.

Introduction

Stem cell transplantation is an innovative treatment strategy that utilizes autologous stem cells to serve as a rescue therapy after high-dose chemotherapy (1). In patients with life-threatening disease, such as hematologic malignancies and inborn errors of metabolism or the immunodeficiency allogeneic stem cell transplantation is a conceivable curative treatment (2). This was before the identification of major histocompatibility complex (MHC), a key feature to success in allogeneic hematopoietic stem cell transplantation (HSCT). Previous results for transplantation were disappointing and no patient survived the treatment (3). The patients were severely ill and succumbed to leukemia or from graft failure (GF), opportunistic infections as well as graft versus host disease (GVHD). Increasing knowledge, technology and experience results have greatly improved (4), leading to important milestones in this area.

The HSCT procedure

When a patient is in need of an allogeneic HSCT a search for a suitable donor is made. The patient and his/her siblings are analyzed with regard to their human leukocyte antigen (HLA) type, the human version of MHC. If no suitable related donor is available a search for an HLA-matched unrelated donor is performed in the international donor registries. The patient then begins the conditioning treatment. The HSCs from the donor (hereafter called the graft) are collected and transported to the patient (recipient). The graft is analyzed, sometimes processed and then administered to the patient as an infusion. Early after the transplantation the patient is isolated until the leukocytes recover. The patient may be treated in reversed isolation in the HSCT ward or be provided conditioning treatment at the hospital followed by a monitored treatment period at home according to the home care program (5–7).

Indications for allogeneic HSCT

The indications for allogeneic HSCT have varied over time due to emerging new treatments. The main indications currently are hematologic malignancies, especially acute myeloid leukemia and acute lymphoid leukemia (8). Other indications involved myelodysplastic syndrome (MDS), hemoglobinopathies (such as thalassemia and sickle cell disease), bone marrow (BM) failure, primary immune deficiencies and inborn errors of metabolism (9).

HLA and transplantation

HLA is basically the human version of MHC. It is inherited and highly diverse, with a wide variety between individuals with its gene location on chromosome 6. They are divided into HLA class I (HLA-A, HLA-B and HLA-C) and class II (HLA-DR, HLA-DP and HLA-DQ). To determine the HLA type of an individual, genotyping using 6-digit high resolution PCR-SSP for both HLA class I (HLA-A, -B and -C) and II antigens (HLA-DRB1, -DQ1 and -DPA) are performed (10,11). To perform an allogeneic HSCT a donor that is HLA-matched on an allele level needs to be identified. According to literature 25% of siblings may statistically be a matched donor and in 30% of all patients a suitable related donor could indeed be found (12). A sibling donor is the first choice and, if possible, a male donor is usually a preferred choice (13). For the remaining two thirds of the patients a donor may be available through the international donor registries. A full HLA match (10/10) is desirable when searching for a donor but some HLA-mismatches may be accepted for certain patients on an individual basis (14,15). Other important factors usually kept in mind included cytomegalovirus (CMV) status, gender, age of donor and recipient and sometimes ABO-blood group. If possible, the best features are a young male donor who is matched for CMV-status and ABO-type. The donor needs to be eligible, i.e., being healthy, tested negative for HIV, HBV, HCV and syphilis and fulfilled the requirements stated above.

Conditioning regimens and immunosuppression

Prior to the transplantation, the patient received a conditioning treatment with cytotoxic drugs and/or total body irradiation. The purpose of the conditioning treatment was to create space for the new marrow cells, eliminate malignant cells and to prevent graft rejection. The conditioning protocol was myeloablative (MAC), constructed to eradicate the recipient BM, or reduced intensity conditioning (RIC). RIC protocols have been observed to cause less organ toxicity thereby less morbidity (16) and also reduced the risk of transplant-related mortality (TRM) (17). There are different RIC protocols with varying MAC effects including non-MAC. The RIC protocols were dependent on the graft versus leukemia (GVL) effect rather than on the chemo- and/or radiation therapies of the conditioning (18,19). In RIC, there is a prolonged period where donor and recipient lymphocytes co-exist with two different antibody-producing immune systems (20).

The choice of conditioning regimen for a patient is based on protocols determined by disease requirements and the patients clinical status. In patients with a malignant disease who received graft from an unrelated donor or umbilical cord (UC) blood anti-thymocyte globulin (ATG) treatment may be added to the conditioning regimen to reduce risk of rejection and prevent GVHD (21). To prevent GVHD after HSCT, immunosuppressive treatment was also given in the form of calcineurin inhibitors such as cyclosporine A or tacrolimus, a short course of methotrexate (a drug that suppresses several cell types within the immune system) (22). The calcineurin inhibitor treatment inhibited T-lymphocyte function and was continued until immune tolerance was achieved; usually 3–6 months post-HSCT, provided that the patient does not show signs of GVHD.

Engraftment and GF

After HSCT the leukocytes from the donor graft recover in the patient and the process is called engraftment (23). Time to engraftment was defined as the first of three consecutive days when an absolute neutrophil count (ANC) in the patients peripheral blood reached ≥0.5×109/l and for platelet (PLT) engraftment the PLT count was ≥50×109/l without platelet transfusions. Primary GF or rejection is defined as BM hypoplasia (<10% cellularity) with a peripheral ANC <0.5×109/l persisting beyond day 21 post-HSCT as confirmed by chimerism analysis with >95% recipient cells. The patients are usually considered to have secondary GF if they initially show signs of engraftment and later develop BM hypoplasia requiring frequent transfusions beyond day 60 after HSCT and no signs of donor cells were detected by chimerism analysis.

Chimerism analysis

To assess the graft function in patients post-HSCT the fraction of donor/recipient origin of white blood cells, chimerism, could be analyzed (24). Chimerism is also used to diagnose early relapse in patients with malignant disease when a reliable disease specific marker is not available (25). Further, signs of recipient type cells (of the same cell lineage as the disease) re-emerging post-HSCT could be an early sign of relapse. So, in this way the chimerism analysis provides important information enabling early therapeutical interventions and thereby better outcome for the patient.

Chimerism analysis of white blood cells is performed on peripheral blood or BM aspirates. Samples are collected from the recipient and donor prior to transplantation and then from the recipient at day 14 after transplantation and onwards according to protocol. After enrichment with immunomagnetic beads (Dynal®), PCR analysis of variable numbers of tandem repeats is used to distinguish donor cells from recipient for T-lymphocytes (CD3), B-lymphocytes (CD19) and myeloid cells (CD33) (26). After 2005, a real-time PCR based on single nucleotide polymorphisms (SNPs) came into play for chimerism (27). Chimerism in the red blood cell population may be assessed by using differences in blood groups between donor and recipient. Prior to HSCT, RBC typing of the donor and the recipient is performed defining a marker, a difference in blood group between donor and recipient. After HSCT, this marker may be exploited to estimate the proportion of donor- or recipient type red blood cells in the recipients blood. This was the routine procedure until the PCR chimerism of white blood cells was introduced.

Complications after allogeneic HSCT

The risk of complications after allogeneic HSCT was dependent largely on the patients immunological status at a particular time-point after HSCT. The main complications after HSCT were infections, GVHD, relapse of the underlying disease and GF/rejection.

The rate of the immunological reconstitution after HSCT is slow and dependent on several factors including age, GVHD, conditioning regimen, graft source and donor (28). For different cell types this period varies considerably (28–30), thus making the patient susceptible to different infectious agents at different times during the post-HSCT period (31). Additionally, even though cell numbers are restored cell function can be impaired for a considerably longer period.

Infectious complications

Barrier defense of the recipient during allogeneic HSCT is primarily responsible for observed complications. Toxic effects of the conditioning regimen disrupt the barriers such as the gut mucosa and skin. This blazes a trail for bacterial and fungal infections, microbes that normally accommodate on the skin and in the gastrointestinal tract, to become invasive and cause disease. The conditioning regimen also often leaves the patient aplastic until the neutrophils recover after 14–28 days post-HSCT (32), i.e., until engraftment. Neutrophils and monocytes are the first cells to recover, closely followed by the NK cells. Consequently, during the first month after HSCT the patient is very susceptible to infections (33).

Both Gram-positive and Gram-negative bacteria, from the skin, mouth and gut pose a problem, as do Candida. For this reason prophylaxis against Candida and bacteria is often given to these patients. The adaptive immunity, T- and B-cells, is in many cases incomplete for several years. The absolute number of T-cells regenerates quite rapidly within the first months after HSCT. However, the T-cell repertoire and function is still impaired for a long time. Early after HSCT memory and effector T-cells derive from mature T-cells originally present in the graft. Thus, the repertoire of antigen specificity of these T-cells is limited to antigens the donor has encountered prior to graft donation. Thus, the quality of the graft is of vital importance. Immunity against new antigens post-HSCT depends on thymic output and the production of de novo T-cells from hematopoiesis post-HSCT. It has been shown that thymic function, measured as T-cell receptor excision circles (TRECs) containing T-cells, deteriorates with increasing age. This could further be influenced by other factors such as graft source [use of peripheral blood stem cell (PBSC)], use of ATG, age and GVHD which all are correlated to decreased TREC levels (34).

Different types of HSCT grafts

In allogeneic HSCT grafts different sources are exploited, commonly PBSCs, BM or UC blood cells (35). In cancer treatments usually PBSC is the preferred choice as it has faster engraftment and offers more GVHD that in turn prevents relapse of the disease (36,37). On the other hand in non-malignant disease, BM graft is is preferred in patients. However, the overall most commonly used graft source today is PBSC.

BM graft. BM contains larger volume, more red blood cells, but less white blood cells and HSCs as compared to PBSC (38). Due to the large amounts of red blood cells in BM, ABO mismatch between donor and recipient have to be considered. In major ABO mismatches the BM may need processing before transplantation. Stimulation of BM donors with G-CSF have been tried to achieve a larger cell dose, thus speeding up engraftment (39).

PBSC. The PBSC collection is performed using aphaeresis technique, most commonly via needles in peripheral veins. The PBSC graft differ slightly from BM grafts not just in blood cell numbers but also in cell composition (40), with T-cells skewed towards Th2 cytokine production, promoted expansion of T regulatory cells, induced interleukin (IL)-4 and −10 production and impaired cytotoxicity of NK cells (41).

UC blood. UC blood is most commonly collected on voluntary basis from UC and placenta after birth. UC can be separated by centrifugation using dextran or HES after collection to reduce volume and deplete contaminating red blood cells (42). The UC blood cells are cryopreserved and kept by UC banks, usually in nitrogen storage tanks. UC was originally mainly used in pediatric patients due to a small total cell dose and their richness in stem cells. However, UC blood cells are an alternative also in adult patients who lacks a suitable related or unrelated donor (43).

Conclusions

The present review concluded that stem cell transplantation is an evolving technique but has associated side effects. Further, research is needed for the pronounced progress of the above therapeutic technique against cancer.