3.4.2 Results

The 3-year overall survival probability (Figure 1) in our cohort was 59%, and a significant difference was found in all three subgroups: CC vs. MC (p < 0.0001); CC vs. microchimerism (p < 0.0001); and MC vs. microchimerism (p = 0.0485). Overall 85 patients died: the main cause of death was a relapse of the primary disease (24%); in the second it was pneumonia (20%); and in the third it was multiple organ failure (15%). Infections, acute GvHD, cerebrovascular accident, other pulmonary complications, and heart attack occurred less frequently. However, if we take into account the distribution of patients in the three subgroups according to their chimerism status, the main cause of death in the CC subgroup was pneumonia (24%), other infections (16%), and multiple organ failure (16%); the relapse was only 5% (two patients). In these patients, the last available sample was taken 2–3 months prior to the relapse date. In the microchimerism subgroup, the main cause of death was relapse (36%), the second most often was multiple organ failure (18%), and in the third, it was pneumonia (14%). In the MC subgroup, most patients died of a relapse (38%), followed by pneumonia (19%), and multiple organ failure (12%).

Overall, the relapse mortality was 67%. In a correlation of 3-year overall relapse rates (Figure 2), there was a significant difference between all subgroups: CC vs. MC patients (p < 0.0001), CC vs. microchimerism patients (p = 0.0073), and MC vs. microchimerism patients (p = 0.0007). Patients with MC relapsed in 72% of cases, and the subsequent mortality was 87%. The detection of MC, especially in the early period after allo-HSCT, is thus an important high-risk factor for a relapse of the disease. In the microchimerism subgroup, patients relapsed in 34% of cases with a mortality rate of 69%. The patients with microchimerism are considered to be potentially at risk. In the CC subgroup, patients relapsed in 15% of cases with a mortality rate of 29%.

Figure 1. Three-year overall survival probability according to chimerism status.

Figure 2. Three-year overall relapse rates according to chimerism status.

#### 4. Discussion

During the last decades, the effect of MC on the occurrence of imminent relapse has been investigated. At the beginning of the monitoring of cell chimerism by RFLP, red cell phenotyping, cytogenetic analysis, or VNTR analysis, there was no correlation found between the presence of MC and a relapse of the primary disease [32, 33]. Following the advances in methodology and the introduction of more sensitive methods for the monitoring of cell chimerism, scientists are increasingly convinced that there is a connection between the presence of MC and the incidence of relapse [34, 35]. These observations also support findings that studied the correlation of MC, minimal residual disease, and the presence of a relapse [36, 37]. Our results support this notion since we found a statistical significant difference between 3-year relapse rates and 3-year survival probability between all three subgroups based on their chimerism status. In addition, there were differences in the main causes of death: patients with MC and microchimerism died most often with a relapse, whereas CC patients died mostly of pneumonia.

In the group of patients with MC, it is also advisable to consider the issue of persisting MC (PMC). In the case of PMC, it depends on the diagnoses. In some diagnoses, PMC is typical and, usually, does not lead to a relapse (e.g., in nonmalignant diseases). In another diagnosis, PMC could be a problem; for instance, in MC patients who have a high percent of autologous cells immediately after the transplantation, it can often lead to a relapse, and the patients are often retransplanted. In our cohort of MC patients, there were eight patients without relapse. One of them relapsed shortly after 3 years, six patients died within first year after allo-HSCT from other causes, and one patient with primary myelofibrosis has never reached CC, and he is still around 1% of autologous hematopoiesis without recurrence of the disease 7 years after allo-HSCT.

The introduction of the RQ-PCR method for cell chimerism level monitoring as a part of routine examination has improved significantly the detection of microchimerism. The early identification of patients at risk is now possible, and, due to the early therapeutic intervention, we can avoid the progression to a highrisk category of MC. Thus, early detection of autologous hematopoiesis is essential for survival. In the case of MC or microchimerism detection, it is necessary to accurately quantify the proportion of recipient genotype and monitor its dynamics over time.

Recently, other studies have also begun to focus on the monitoring of cell chimerism using the latest technologies such as digital PCR [38] or next-generation sequencing [39]. The incorporation of these techniques into routine investigations depends on subsequent validations that will reveal their advantages or disadvantages.
