Over the last 30 years, allo-HSCT has become a standard treatment for many hematological malignancies and is now increasingly used also as immunotherapy for the treatment of solid tumors [1,2]. During allo-HSCT, the recipient receives a preparative conditioning regimen (e.g., chemotherapy, radiotherapy) to destroy his hematopoiesis and immune system. This practice is followed by the administration of Hematopoietic Stem Cells (HSC) harvested from the donor. The donor’s HSC engraft, proliferate and finally reconstitute hematopoiesis in the recipient [3,4]. The result is the creation of a biological chimera, a term used to describe the presence of tissues of different genetic origin in the same organism; the hematopoietic cells derive from the donor, while the other tissues (e.g., epithelium) are genetically derived from the patient-recipient. This unphysiological formation of biological chimeras is not free of consequences. After allo-HSCT, epithelial tissues become injured through the preparative regimen and are then potentially attacked by allo-reactive T cells. The net effect of these allo-antigeneic reactions is tissue stress and apoptosis, which we recognize clinically as Graft-versus-host Disease (GvHD) [5].

DNA is a reactive molecule and is constantly attacked and modified by external and internal agents. A mammalian DNA molecule can undergo about 100,000 modifications per day and replicates with an error rate of one error per 10¹⁰ nucleotides [6]. Genome integrity is a basic element of cellular homeostasis. To maintain genome integrity and ensure its stable inheritance during replication, cells are equipped with several mechanisms, including DNA repair, cell-cycle checkpoints and programmed cell death [7]. The term genomic instability (GI) describes the failure to transmit an accurate copy of the entire genome from one cell to its two daughter cells. The existence of genomic instability is a sign of the failure of the protection mechanisms and has been correlated with cancer and cancerous transformation [8].

Microsatellites are short tandem repeat sequences (repeat units range from 1–6 bp in length) dispersed throughout the genome. There are more than 1 million microsatellite loci in the human genome, which comprises approximately 3% of the genome. Microsatellite sequences are among the most variable types of DNA sequence in the genome. Their polymorphism derives mainly from variability in length, rather than in primary sequence. Though highly polymorphic, as their length varies in a population, they are inherited stably and are unique to each individual, which means that the length of a microsatellite is the same in all the cells of the same person [9]. For this reason, the detection of different MS polymorphisms with PCR-based assays can be used for linkage mapping, paternity testing and forensic purposes, as well as for chimerism quantification after allo-HSCT [9,10]. The term microsatellite instability (MSI) describes alterations in the length of a microsatellite locus detected by PCR amplification of an individual’s DNA [7]. Such length alterations could refer to insertion or deletions of repetitive units, which may be the result of polymerase slippage during DNA replication [11]. The repetitive nature of microsatellites makes them especially prone to such damage [11,12]. According to several studies, there are indications that the DNA structure of these repetitive sequences is not in the normal B-conformation in vivo and, therefore, binds poorly to DNA repair enzymes [13], which makes it susceptible to DNA damage. Therefore, the detection of MSI indicates a general GI, where mutations in DNA can be accumulated and which may be related to carcinogenesis [9,14]. MSI was first detected and described in individuals with a type of hereditary colon cancer (HNPCC, hereditary non-polyposis colorectal carcinoma), which is caused by mutation of the DNA-mismatch-repair gene MSH2[14]. In addition to HNPCC, MSI has also been reported in several sporadic cancers (colorectal, bladder, skin, lung and ovarian) [14–17]. Recently, MSI has also been observed in chronic inflammatory diseases, such as ulcerative colitis and rheumatoid arthritis [18,19]. The exact mechanisms through which chronic inflammation may lead to these genomic alterations are the subject of continuing debate. The biologic significance of genomic instability in chronic inflammation settings is yet unknown. In ulcerative colitis, MSI has been associated with increased cancer risk [20], while there is no such indication in rheumatoid arthritis [19].

Faber et al.[21] hypothesized that the chronic tissue stress resulting after allo-HCT may cause genomic alterations in host epithelium. The donor-derived lymphocytes may interact with and damage recipient epithelial cells at the molecular level in these chimeric individuals. Examination of tissues isolated from patients treated with allo-HCT revealed frequent genomic alterations, which were detected as microsattelite instability (MSI). These genomic alterations were found only in allogeneic transplanted patients, but not after autologous HSCT or intensive chemotherapy, and therefore, they suggested that factors implicated in the alloreactive microenvironment after allo-HSCT are substantially involved in the mutation process. Further analyses performed by our group [22] in patients who underwent allo-HSCT also confirmed the presence of frequent MSI in the non-neoplastic tissues of allogeneic transplanted patients. This was most frequently found in older patients, in males transplanted with cells from female donors and in patients that experienced an extreme GvH reaction. In an in vitro mutation analysis system, we found that allostimulated mixed lymphocyte cultures may induce MSI in co-cultured epithelial cells [22]. These results were independently confirmed by subsequent studies from other groups, which reported that the detection of MSI [23] and chromosomal instability [24] in the epithelial tissues of patients after allo-HSCT was correlated to the presence of GvHD inflammation.

Replication errors during DNA synthesis occur with a defined probability in all cells and may result in a change in the length of microsatellite loci. The mismatch repair (MMR) system recognizes and corrects these genomic alterations [6]. Whenever a DNA error cannot be repaired, apoptosis pathways are activated [7]. Thus, cells with microsatellite alterations, which escaped repair, will not replicate and, therefore, in molecular studies, should not become apparent among the large excess of the surrounding normal cells [25]. However, if deficient DNA repair is coupled with a failure to elicit an apoptotic response, this association may result in a growth advantage sufficient to generate a detectable clonal population of cells that share genetic alterations. Therefore, the detection of MSI in clinical samples indicates the presence of a cell population, which (i) was exposed to a factor that caused MSI, (ii) failed to repair the genetic damage through normal DNA repair mechanisms, (iii) were not eliminated by apoptosis via activation of DNA-damage checkpoints and finally (iv) multiplied, and perhaps acquired, a growth advantage so as to create a detectable clonal population (Figure 1).

MSI has been shown to be an indicator of genomic instability [9]. It is very likely that mutations occur not only in non-transcribed MS, but also in coding regions of the genome [57]. Mutations in coding genes might be responsible for the protean post-transplant GvHD-related clinical syndromes and phenotypes, such as scleroderma, Sjögren syndrome, musculoskeletal or pulmonary disease and many others. In addition to serving as an indicator for genomic instability, mutations of microsatellites may directly contribute to evolution of post-transplant diseases. Although microsatellites are thought to be part of the “junk” DNA dispersed in non-coding regions throughout the genome, there is now growing evidence that non-coding DNA distribution is not random. As shown by the accumulating data of the ENCODE project, the previously thought to be “useless” DNA in fact contains functional elements rich in protein and RNA binding sites and brings them to the ideal conformation to regulate the function and expression of protein-coding genes [58]. Therefore, it seems that a potential change of a microsatellite length could end up in possible relevant changes of the cellular phenotype. In addition, it is already known that there are microsatellite sequences within genes, which play a regulatory role in gene expression [11]. TGFRII, IGFIIR and BAX genes are paradigms of transcribed genes, which contain microsatellites within their coding regions [14,57]. Changes in the lengths of these repetitive sequences, when found within coding regions of specific genes, have been shown to result in gene inactivation or modification of function of the gene and cause disease in humans such as Huntington’s disease and myotonic dystrophy [57].

The detection of MSI in clinical samples has been mainly associated with cancer [14] or cancer risk in chronic inflammatory diseases [20]. In hereditary nonpolyposis colorectal carcinoma, the MMR deficiency plays an aetiological role in the development of MSI and carcinogenesis [14]. Microsatellite alterations are also common in various sporadic cancers, such squamous cancers [14]. It is interesting that squamous cell carcinomas are the most common secondary tumors after allogeneic HSCT [59] and are associated with chronic alloantigeneic stimulation through GvHD. Statistical analysis of clinical data in a recent study by our group showed a significant correlation of MSI detection in the epithelium of allotransplanted patients and the development of secondary squamous cell neoplasia. This observation enhances the possibility of a pathogenetic role of MSI in secondary malignancy development. Deeg et al. found in eight post-transplant tumors a p53 expression indicative for mutations of the p53 gene [54]. Themeli et al. [22] found p53 mutations in two out of four post-transplant tumors, but no p53 mutations in six samples where MSI was detected. These findings indicate that MSI occurrence is an early phenomenon after allo-HSCT. Therefore, the hypothesis that MSI may characterize a precancerous state of the epithelial cell seems logical. The mutator phenotype may further contribute to the development of new mutations in coding regions with important function, such as p53 gene, and promote carcinogenesis. Genome-wide analyses in allografted recipients may indeed identify specific genomic alterations, which might be responsible for or used as molecular biomarkers of post-transplant diseases, including secondary cancer.

Taken together, it seems that in the GvHD tissue environment cytokines, ROS and NO induce genomic alterations in epithelium by acting through several different, but communicating, pathways. The oxidative stress damages the DNA, causing mutations, such as microsatellites length alterations. The induction of mutations is facilitated by a relaxation of DNA repair system mediated by NO or ROS. The cells may survive due to a possible inactivation of the DNA-checkpoint apoptotic mechanisms. Furthermore, the donor-derived immune system in an allogeneic setting may attack the recipient-derived normal epithelial cells, yet spare cells with an MSI phenotype, providing in this way a selective growth advantage for the cells with novel repeat lengths (Figure 3). Elucidating the ultimate mechanisms underlying the genomic instability following alloantigeneic reactions in the chimeric organism is a major challenge. Findings may provide more information about GvHD pathology and pathogenesis of post transplantation clinical outcomes, such as secondary malignancies and GvHD related syndromes. Focusing on the pathways through which the alloantigeneic reaction causes genomic instability may bring up novel therapeutic targets for the protection of the epithelium during GvHD and the prevention of malignant transformation.