Overcoming limitations in the detection of mixed chimerism

This paper highlights the importance of chimerism as a diagnostic tool for clinicians treating transplanted patients. Furthermore, the evolution of novel diagnostic tools for early detection of mixed chimersim is discussed. The recent development of NGS technology offers the possibility to analyse mixed chimerism with both sensitivity, as well as accurate and precise determination.

Flow Cytometry, restriction fragment length polymorphism (RFLP), PCR of Short Tandem Repeats, qPCR and Next Generation Sequencing are examined as potential methods for detecting mixed chimerism. 

The term chimera derives from Greek mythology and was first described by Homer in the Iliad as a firebreathingmonster in Asia Minor, composed from parts of multiple animals. Chimerism is defined as the presence of cells or tissues originating from another individual than the host derived cells.

This situation can occur naturally during pregnancy: where fetal cells circulate within the maternal blood stream, in dizygotic twin pregnancies with separate placentas, or after transplantation. The fact that cells from two (or more) genetically separate individuals can co-exist within one body has led to development of new techniques to discriminate the amount of the two genetic individuals within the organism.

While chimerism is to be expected with any transplantation, the amount of chimerism can differ in different transplant settings. For instance, solid organ transplantation means transferring tissues or solid organs into the host with little amounts of circulating donor cells within the blood stream. In contrast, hematopoetic stem cell transplantation (HSCT) results in continous recirculation of donor cells within the host. These facts have led to the evolution of different laboratory techniques to define the amount of donor DNA or cells within the host. One important reason for monitoring of patients post HSCT is to allow the earliest possible medical intervention and best possible patient outcomes.

Flow Cytometry, restriction fragment length polymorphism (RFLP), PCR of Short Tandem Repeats, qPCR and Next Generation Sequencing are examined as potential methods for detecting mixed chimerism. 

The paper is authored by Dr. Dan Hauzenberger, Medical Director of the Section for Transplantation Immunology at Karolinska University Hospital, Sweden. 

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Overcoming limitations - mixed chimerism - Devyser ER08 - Figure 2
Figure 2. Principle of chimerism analysis using STR and Q-PCR techniques (adopted from Uzunel et al. 2003)

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The importance of detecting mixed chimerism

The introduction of hematopoietic stem cells (HSC) from bone marrow (BM) or peripheral blood (PB) as a curative treatment for patients with malignant or non-malignant haematological diseases has been one of the major medical advancements of the last 30 years. In 1957 the first attempt at performing bone marrow transplantation was made in several patients suffering from malignant hematological diseases (Thomas, Lochte et al. 1957).

Early attempts at using hematopoietic allogeneic stem cell transplantation (HSCT) for treatment were however poor with many patients dying in complications directly related to the transplantation. However, with increasing knowledge of the importance of the polymorphic HLA system (Human Leucocyte Antigens) and immunosuppression, results of transplantations improved and are today the only curative treatment for patients with malignant or non-malignant haematological diseases (Ringden, Groth et al. 1995, Ringden, Lonnqvist et al. 1995).

Today more than 50 000 patients annually undergo HSCT world-wide. The majority of these patients have an underlying malignant disease such as acute myeloid leukemia (AML), acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia in blast phase, Myelodysplastic Syndrome (MDS), multiple myeloma, high-risk lymphomas and Hodgkin’s disease. Furthermore, several non-malignant diseases can also be treated successfully using HSCT. Among them are several immunodeficiencies, such as severe combined immunodeficiency (SCID), Wiskott-Aldrich syndrome, and common variable immunodeficiency (CVI) (Saba and Flaig 2002).

At least four different clinical complications following HSCT have been described; toxicity related to the pre-treatment, infections, recurrence of the underlying malignant disease and immunological reactions including graft-versus-host-disease (GVHD) (Barrett, Horowitz et al. 1989, Horowitz, Gale et al. 1990).

Recurrence of the underlying malignant disease (relapse) is the most frequent cause of treatment failure in patients undergoing HSCT for leukaemia. Relapse rates of up to 20% have been reported in patients who received transplants in the early stages of their disease. Patients with more advanced diseases show higher incidence of relapse, reaching 50-70% in some reports, with T cell depletion and absence of GVHD as the most important risk factors (Horowitz, Gale et al. 1990, Marmont, Horowitz et al. 1991).

Leukemic relapse occurs, in general, in the recipient-derived cells due to incomplete eradication of the malignant clone, poor graft-versus-leukaemia (GVL) effect or de-novo malignant transformation following treatment with oncogenic substances. Treatment of malignant relapse can be performed in several ways including cyclosporine discontinuation, chemotherapy, second allograft, G-CSF or donorlymphocyte infusions (Kolb and Bender-Gotze 1990). Therefore, in addition to treatment strategies for leukemic relapse, development of novel techniques for early detection of relapse has been of major importance for these patients. ”..development of novel techniques for early detection of relapse has been of major importance for these patients”

OVERCOMING LIMITATIONS IN THE DETECTION OF MIXED CHIMERISM 

The main aim of HSCT in patients with malignant diseases is, as mentioned, the eradication of the malignant cell clone. Since the complete eradication of the malignant cells is difficult to measure, complete remission (CR) has been used for defining successfully treated patients. CR in acute leukaemias is in general defined as; QQ Bone marrow blasts <5%; QQ absence of circulating blasts; QQ absence of extramedullary disease and recovering; or QQ normalized peripheral blood counts.

Determination of complete remission in patients undergoing HSCT has mainly been based on counting cells in blood and bone marrow using light microscopy or flow cytometry. The presence of minimal numbers of detected or non-detected malignant cells in blood or bone marrow has been termed minimal residual disease (MRD) (Lion, Daxberger et al. 2001, Uzunel, Jaksch et al. 2003). Success in treatment intervention is very much dependent on the availability of diagnostic techniques for early (i.e. sensitive) detection and quantification of minimal residual disease. Early treatment made possible by more sensitive diagnostic methods is expected to improve patient survival and significantly reduce the costs for management of transplant patients.

With the introduction of novel molecular as well as non-molecular techniques with higher sensitivity, the limit of detection of remaining malignant cell clones has vastly improved. An overview of the techniques available for detection of minimal residual disease and their limit of detection is shown in figure 1.

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