Patients and methods

Patients

Chimerism analysis was performed in blood of 54 matched related allogeneic transplant patients. All patients were given myeloablative conditioning. In total, 46 patients received peripheral blood stem cells (PBSC) and eight patients were recipients of BM. The conditioning regimen was TBI/cyclophosphamide for acute lymphoblastic leukemia patients and busulfan/cyclophosphamide for the rest of the patients. All PBSC donors were mobilized with G-CSF (10 g/kg/day). Collection of PBSC was performed with a Baxter Fenwall 3000+ CS cell separator 12 h after 4th and 5th G-CSF dose. BM was collected according to the center's established procedure in the operating theater under general anesthesia. PBSCT patients received median number of 6.3 10⁶ CD 34+ cells per kilogram of body weight (range 2.8–8.6), while BM recipients were infused median number of 3.2 10⁶ CD 34+ per kilogram of body weight (range 1.1–5.2).

Patients' and donors' characteristics and indications for transplantation are shown in Table 1.

Table 1 - Patient and donor characteristics.

Full table

Primary graft-versus-host prophylaxis consisted of cyclosporine and methotrexate according to the Seattle protocol.⁵

Graft-versus-host disease

Acute and chronic graft-versus-host disease symptoms were evaluated and graded according to the Seattle criteria.^{6, 7}

Chimerism assessment

Chimerism was assayed on days 30, 60, 100 and 180 post transplant and then every 3 months. The median period of chimerism evaluation was 22 months (range 13–51). White blood cell DNA was extracted with a commercially available kit, Blood DNA Prep Plus (A&A Biotechnology, Poland), and DNA from particular subtypes of WBCs was extracted with a kit for biologic evidence testing Sherlock AX (A&A Biotechnology, Poland). The amount of DNA was measured with fluorometry. DNA samples were amplified (multiplex PCR) with a commercially available Profiler Plus (Perkin Elmer, USA) kit consisting of nine STR loci (D3S1358, VWA, FGA, D8S1179, D21S11, D18S51, D5S818, D13S317 and D7S820) as well as a sex marker (amlogenin locus). DNA amplification was carried out according to the manufacturer's conditions on 2400 Perkin-Elmer thermocycler. For each amplification optimal DNA concentration was used (1.25 ng DNA/25 l of PCR mixture). The size of fluorescent stained PCR products was analyzed by capillary electrophoresis on an automatic sequencer DNA ABI310 (Perkin-Elmer, USA). PCR products were fractionated in a 47 cm length capillary tube filled with polymer POP4. Electrophoresis was performed at constant voltage for 24 minutes. The internal DNA size standard was ILS400 marker labelled with CRX (Promega, USA). The analysis of DNA fragment size was carried out with GeneScan v 2.0 software (Perkin-Elmer). The size of amplified allele was compared to a standard in 'allele ladder' included in the set. It consisted of all sequenced alleles for the loci in Profiler Plus.

The genetic profile of each recipient/donor pair was assessed before transplantation in a sample of white blood cells. In most cases the number of informative loci was two per patient and only in eight cases were three loci regarded as informative. Those loci which were most different in the recipient and donor and in which no allele was shared by both recipient and donor or presented as a 'stutter' (n=4) were used for further analyses. In such a case, chimerism was evaluated according to the formula:

In this formula, R1 and R2 represent areas under the curve of particular recipient allele peaks, while D1 and D2 represent area under curve of particular donor allele peaks.

In a case when within tested locus heterozygous donor and recipient had a common allele chimerism was evaluated according to the formula:

In this formula, R1 represents the area under the curve of recipient allele peak and D2 represents area under curve of donor allele peak. Loci chosen at initial analysis were used throughout chimerism monitoring.

Additionally, 15 of examined patients had chimerism evaluated in the subsets of white blood cells: T lymphocytes, NK cells, monocytes and granulocytes.

Complete chimerism was defined as presence of donor only DNA in tested samples. Any amount of recipient DNA in the samples was classified as MC.

Isolation of leukocytes subsets

Leukocyte subsets were isolated after monoclonal antibody staining (anti CD3, CD19, CD16/CD56, CD14 and anti CD66b) with FACS Calibur Becton Dickinson flow cytometer equipped with cell sorter and concentrator. Granulocytes CD66b staining was performed in the whole blood. To enhance purity and recovery of other cell subsets, staining and sorting was carried out on isolated peripheral blood mononuclear cells. B-lymphocytes constituted a minor fraction of WBC until day 180 after transplantation and it was impossible to have them sorted as a representative population for further analysis.

Each sorted population consisted of at least 50 000 cells. The purity of the sorted population was checked every time with flow cytometry. DNA analysis was performed only if the purity of the cell population exceeded 97%.

Statistics

The ² test was used for statistical calculations. Correlation between time of CC and difference between CC and cGvHD development was evaluated with Pearson's correlation test.

Results

Chimerism

All 54 patients were tested for hematopoietic chimerism in unselected white blood cells. Of those patients, 25 achieved complete hematopoietic chimerism in the peripheral blood within 100 days after transplantation (early CC). Only nine patients showed CC as early as day 30. Another 10 patients showed CC at day 60 and six patients showed CC at day 100. Median time to achieve CC in this group was 60 days. CC was achieved in 26 patients in the course of immunosupression taper or after withdrawal (late CC – later than day 100 post transplant). Median time to CC in this group was 270 days (range 180–600). All patients in this group showed no more than 2% self-DNA in the period between 100 days post transplant and CC. In three patients, persistent continuous MC was noted throughout the period of observation. According to the aim of this analysis, those patients were excluded from further considerations.

Graft-versus-host disease

Clinically important (grade II or more) acute graft-versus-host disease was diagnosed in 42 and 38% of patients in early and late CC groups, respectively. Frequency of grade III and IV aGvHD was also similar in both of them.

Chronic GvHD was diagnosed in 70% of analyzed patients (36 of 51). Of 25 patients with early CC, 23 developed cGvHD. In 21 of those patients cGvHD had an extensive type characteristics. In patients with late CC extensive type cGvHD developed in eight of 26 subjects (Figure 1). The difference in extensive cGvHD frequency in early and late CC groups was statistically significant (P<0.001). Also, 'de novo' onset of extensive cGvHD was more frequent among early CC patients although the difference was not statistically significant (P=0.066). The severity of cGvHD course was reflected by the number of immunosuppressants necessary to control the disease. In the group of early CC more patients needed three or more drugs (12 of 23) than in late CC group (two of 13). The difference was statistically significant (P<0.005). In early CC group, three patients died of cGvHD progression, while in late CC group no such death was noted. In the group of patients with early CC symptoms of cGvHD were uniformly preceded by CC achievement (mean time from CC to cGvHD was 85 days). On the other hand, symptoms of cGvHD in patients who achieved late CC usually preceded CC achievement (mean time from cGvHD to CC was 100 days). In only three cases of this group was CC obtained a short time before appearance of cGvHD symptoms. Correlation between time of CC and the difference in time of CC and time of cGvHD emergence was statistically significant (P<0.001) (Figure 2). Characteristics of cGvHD in the groups of early and late CC are presented in Table 2.

Figure 1.

Chronic GvHD type in late and early chimerism groups.

Full figure and legend (48K)

Figure 2.

Correlation of time to CC after transplant and the time difference between CC and cGvHD diagnosis.

Full figure and legend (18K)

Table 2 - Incidence and characteristics of cGvHD in early and late CC groups.

Full table

Relapse

Relapse rates in early and late CC groups were not statistically significant (three and five patients respectively).

Subpopulation chimerism and cGvHD

In 15 patients more detailed analysis was performed. Chimerism was evaluated in particular white blood cell subset: T lymphocytes, NK cells, monocytes and granulocytes. Of those patients 12 were subjects of further analysis (those achieving CC in the peripheral blood at any time of observation). Three patients were excluded due to persistent MC.

Of 12 analyzed patients, three achieved CC in WBC before day 100 post transplant and nine patients achieved CC in WBC during the course of further observation. However, eight of those 12 patients expressed MC in at least one subset of WBC in spite of showing CC in the whole white blood cell population.

Most patients showed early complete chimerism in the population of monocytes (CD14), granulocytes (CD66b) and NK cells (CD3^-CD16/56⁺). On day 30 post transplant 92, 85 and 85% of patients showed CC in those cell subset, respectively.

Complete hematopoietic chimerism in the subset of T lymphocytes was detected in three subjects (23%) before day 100. Those patients showed also CC in unselected WBC of the peripheral blood. All three patients developed extensive cGvHD (100%). One patient achieved CC in both WBC and all tested subsets at day 30 post transplant and developed progressive-type cGvHD preceded by acute GvHD grade III and needed three-drug immunosuppressive treatment.

In nine patients who achieved CC in WBC after day 100 post transplant, the predominant persistent population with MC was T cells (Figure 3). Additionally, one patient showed MC in NK cells and one in both granulocytes and NK cells. In this group of patients, cGvHD was noted in six patients, three of whom developed extensive cGvHD (33.3%). Two patients show persistent MC in CD3 subset of peripheral blood cells while in unselected WBC CC is detected. Those patients never developed symptoms of cGvHD. Those are patients with chronic myeloid leukemia who never achieved molecular remission of the disease after transplantation and present constant BCR/ABL expression in the absence of Ph chromosome either by conventional cytogenetics or FISH (both more than 24 months in observation).

Figure 3.

Example of mixed chimerism in two loci within CD3 lymphocytes (vertical arrows) in multiplex STR-PCR assay.

Full figure and legend (49K)

Discussion

The use of most methods of chimerism evaluation leads to complete chimerism detection as early as day 30 post transplant.^{1, 2, 8, 9, 10, 11, 12} Employment of a more sensitive method (multiplex STR-PCR assay) and strict definition of complete hematopoietic chimerism enabled us to distinguish two groups of patients. One group of patients achieved true CC by day 100 after transplantation. In this group elimination of recipient hematopoiesis occurred while all patients were still on full GvHD prophylaxis (early CC). The other group achieved CC at a later time in the course of immunosuppressive therapy taper or after withdrawal (late CC). The definition of complete chimerism differs among authors who are interested in this particular field. Many of them define chimerism as complete if 95–98% of donor DNA is detected.^{1, 2, 8, 9} That leaves 2% of host DNA, which may be composed of DNA derived from immunologically important cells responsible for various reactions like disease control or immunological tolerance induction and prevention of GvHD development.^{13, 14, 15, 16} Therefore, our strict definition of complete chimerism as total absence of detectable host DNA by a very sensitive method sets a precise threshold for clinical considerations. Particularly that in all our patients who achieved late CC (more than 100 days post transplant) amount of self-DNA in the period of MC never exceeded 2%.

An interesting observation is that from the clinical point of view we were able to distinguish two well-defined groups of patients as to the time of cGvHD emergence and its severity. In the first group median time to cGvHD symptoms was 131 days while in the other group it was 270 days after transplantation. Moreover, the first group consisted uniformly of patients who achieved early CC (at a median time 60 days post transplant) and the mean time from CC to cGvHD was 85 days and most patients developed extensive disease. In all those patients CC was achieved before symptoms of cGvHD appeared. In the other group median time from transplantation to cGvHD was 250 days. Those patients achieved CC at a median time of 270 days after transplantation. In all but three cases in this group achievement of complete chimerism was preceded by cGvHD symptoms and mean time from cGvHD to complete donor chimerism was 100 days. In that group less cGvHD was diagnosed and the disease in general was not as severe as in the first group. As a surrogate marker of cGvHD severity was the difference in immunosuppressive treatment requirements between the groups. More patients in the early CC group needed three or more drugs than in the late CC group. The difference was statistically significant.

The mechanisms that may lead to such a heterogeneous pattern of CC achievement and development of chronic GvHD still remain obscure. It may be speculated that the mechanisms leading to CC achievement and emergence of cGvHD in the two groups are different. In both groups of our patients incidence of clinically evident acute GvHD was similar. In the group with early CC chronic GvHD was in most cases diagnosed as a 'de novo' disease and in all but two cases the type of the disease was extensive (21–25). It may suggest that CC achievement is not directly linked to preceding acute graft-versus-host disease. However, it is impossible to exclude existing subclinical graft-versus-host reaction leading to elimination of host cells.¹⁷ In the light of recent papers on transplantation immunology also dealing with organ transplants, the concept of microchimerism may to some extent explain different pattern of CC achievement and cGvHD observed in our patients. Coexistence of host and donor cells in transplant recipients leads to easier induction of immunological tolerance through a peripheral mechanism.^{18, 19} In hematopoietic stem cell recipients it may express clinically as a lack or mild course of chronic graft-versus-host disease. It seems that the most critical period with great influence on the whole post transplant course is the first 3 months after transplantation. Elimination of all detectable host cells during the early period in which the patients receive standard full-dose immunosupression may be an indirect evidence of intense alloreactivity of donor cells. As a consequence, a protective tolerance-inducting clone is also eliminated leading to a more severe cGvHD. This may be confirmed by clinical observations and chimerism evaluation in our patients with early CC. Some authors indicate that the cell population responsible for tolerance induction is CD3 lymphocytes.^{20, 21, 22} Also, some investigators stress the influence of CD3 MC on relapse risk even though persistent population is not of malignant clone.^{22, 23, 24, 25} In our observations, patients who retained MC for longer period (late CC) developed symptoms of cGvHD later and of lesser severity. In all those patients, chimerism assessment in subpopulations showed that cells responsible for MC were CD3 lymphocytes. In this group of patients, elimination of this 'protective' clone can be explained as a result of chronic GvHD, which develops in the course of tapering or after withdrawal of immunosupression. Acquired tolerance during CD3 MC may lead to less and a milder course of cGvHD. On the other hand, elimination of CD3 lymphocytes within the first 3 months after transplantation like in our early CC group results in statistically more frequent extensive cGvHD, which requires more intensive treatment. Our results were obtained from a relatively small group of patients and therefore investigation of a larger population is advisable.

References

References

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Acknowledgements

The research was supported by grant from the State Committee of Scientific Research 4PO5BO4419 (Poland).