Abstract

The molecular analysis of individual hematopoietic chimerism at a defined time after allogeneic hematopoietic stem cell transplantation represents an important non-specific marker of posttransplant course. The monitoring of its dynamic allows the identification of patients at a high risk of relapse. A variety of methods are used for the monitoring of cell chimerism. It is necessary to use sensitive molecular genetic methods for early detection of the autologous hematopoiesis. Quantitative multiplex real-time polymerase chain reaction (PCR) analysis can serve as a very sensitive (0.01–0.1%), relatively quick, and inexpensive method to detect <1% of minor genotype. With an increasing ratio of minor genotype (>1%), it is more suitable to use short tandem repeats (STRs) for its analysis. Based on the differences in recipient/donor pair genotypes, at least two suitable informative polymorphisms located at different chromosomes can be selected. The combination of methods is appropriate, and the choice of the used method depends on the patient's actual chimerism status. The cohort of 207 patients monitored at the Institute of Hematology and Blood Transfusion was divided into three subgroups according to their chimerism status (complete chimerism (CC), microchimerism, mixed chimerism (MC)) 3 years after allogeneic hematopoietic stem cell transplantation (allo-HSCT). A significant difference in the 3-year survival and 3-year relapse rates in all three subgroups was found.

Keywords: chimerism, hematopoietic stem cell transplantation, real-time polymerase chain reaction, short tandem repeats

### 1. Introduction

The term chimerism comes from Greek mythology. A chimera is a creature with a lion's head, a goat's body, and a snake's tail. In biology, it is a unique state where cells from genetically different individuals coexist within one body [1].

A chimerism can spontaneously occur in several situations; probably the most common is the persistence of fetal progenitor cells in maternal blood [2]. On rare occasions, a chimerism can be developed in the uterus through the fusion of two genetically different zygotes (so-called tetragametic chimerism) or from a stem cell transfusion between dissimilar fraternal twins that share a placenta [3].

The latter possibility is an engraftment of maternal hematopoietic stem cells, especially in children with severe combined immunodeficiency.

Artificial cell chimerism can arise after transplantation of an organ, tissue, or hematopoietic stem cells (HSCT). The allogeneic HSCT (allo-HSCT) is one of the most used curative options for the treatment of hematological malignant and nonmalignant diseases, and for some diagnoses, it is currently the only available therapy. The long-term success of allo-HSCT depends on many factors such as an appropriate condition regimen (which destroys all leukemic cells), the state of patient in the time of HSCT (active disease vs. disease remission), the donor's age (T lymphocytes of younger donors are more willing to activate and destroy leukemic cells), the modification of the graft (T-cell-depleted graft vs. unmanipulated graft), and engraftment dynamic of HSC (it was proved that early achievement of full donor chimerism predicts lower relapse risk in acute lymphoblastic leukemia (ALL) patients) [4].

The analysis of cell chimerism is an integral part of the posttransplant monitoring of patients. In the immediate posttransplant period, this determination enables the identification of engraftment dynamics or graft failure, and it allows the early detection of a patient's increasing hematopoiesis which indicates a disease relapse.

The principle of the examination is based on the fact that each person has a unique DNA structure that comprises a set of highly variable polymorphisms; most polymorphisms are found in the noncoding regions of the genome [5, 6]. Thus, a comparison of the variable DNA polymorphisms of a patient and a donor provides information about the origin of the cells in almost every case.

### 2. Methods for the monitoring of cell chimerism

There are a number of molecular methods (e.g., sex-specific markers, fluorescence in situ hybridization (FISH), cytogenetic methods, erythrocyte phenotyping) that have been used for some time for the monitoring of cell chimerism; however, all of these techniques have their own limitations. They are laborious or time-consuming as well as have low informativity and sensitivity, (for a review, see [7, 8]).

#### 2.1 History of molecular genetic methods

The first method of DNA analysis to take advantage of sequence polymorphisms was the method of restriction fragment length polymorphism (RFLP) where genomic DNA is digested with restriction endonucleases (restrictases) [9]. The size of the generated fragments is individually specific and depends on the various numbers of repetitive units in different individuals. All restriction fragments can be subsequently separated by gel electrophoresis. Southern blotting is used to transfer DNA from gel to filter membrane. The fragments are then detected by probe hybridization. For RFLP analyses it is necessary to extract high molecular weight undegraded genomic DNA.

The revolution in the monitoring cell of chimerism arose with the introduction of polymerase chain reaction (PCR) [10]. The first historical method based on PCR is amplified fragment length polymorphism (AFLP) [11]. Genomic DNA is digested by restriction enzymes, and the adaptors are subsequently ligated to the restriction fragments followed by selective PCR amplification with primers compatible to the adaptor's sequence. The amplicons are then separated by gel electrophoresis.

In laboratory practice, the most common current methods for long-term monitoring of cell chimerism are summarized in Table 1. They are based on the


Suitable Molecular Genetic Methods for the Monitoring of Cell Chimerism DOI: http://dx.doi.org/10.5772/intechopen.88436

VNTR, variable number of tandem repeats; STR, short tandem repeats; InDels, short insertions and deletions; SNP, single-nucleotide polymorphism; PCR, polymerase chain reaction.

#### Table 1.

Current possibilities of polymorphism analysis.

genotyping and quantification of different polymorphisms using PCR which provides an unlimited number of copies of a specific DNA segment based on a single copy.
