**3. SNP and NP DNA markers in chimerism monitoring by 'RQ PCR'**

#### **3.1 Introduction**

Chimerism monitoring allows the characterisation of the haematopoietic stem cell origin in the recipient's blood or bone marrow after allo-hSCT. The DNA identification of the person is mainly based on the DNA polymorphisms, which include singlenucleotide polymorphisms (SNPs) due to nucleotide substitutions and insertion or deletion of one or more nucleotides (indels). The late can be multiallelic (STR) and diallelic (biallelic). The extensive bibliographical search for human diallelic indels and its basic properties were determined and selected for chimerism monitoring [13, 38]. The sensitive quantitative real-time PCR analysis using indel polymorphisms can be the useful tool to predict relapse in leukaemia patients after allo-hSCT [39].

#### **3.2 Complete and mixed chimerism monitoring by SYBR green-based real-time PCR method ('RQ PCR')**

Tens of biallelic (diallelic) nucleotide polymorphisms specific for the donor and the recipient can be detected by the method of allele-specific real-time PCR.

#### *3.2.1 Cohort*

The cohort consisted of 188 patients from University Hospital Bratislava, Slovak Republic and 188 donors (111 from relatives and 77 from national and world registers of the bone marrow).

#### *3.2.2 Methods*

The DNA was isolated (NucleoSpin Blood, Macherey-Nagel) from peripheral blood leukocytes (centrifugation 3000 × g/10 min) before allo-hSCT from both donor and recipient and 30, 100, 180 and 365 days after allo-hSCT only from recipient (patient). Concentration of the DNA was measured on NanoPhotometer, Implen and samples were diluted to 10 ng/μl. To get allogenic and autologic informative markers for quantification after allo-hSCT, screening of donor and recipient DNA samples before allo-hSCT was essential. Three 1 μM allele-specific primers (two forward and one reverse or one forward and two reverse) for 11 biallelic nucleotide polymorphic markers were localised on 1st, 5th, 6th, 9th, 11th, 17th, 18th, 20th and X chromosome. Also, two pairs of specific primers (forward and reverse) for monoallelic DNA marker on Y chromosome (amelogenin) and for endogenic control gene GAPDH were used (**Figure 4**) [40]. The amounts for one-well allele-specific PCR reaction on 96-well plate were: 12.5 μl Power SYBR Green (PCR Master mix, Power SYBR green; Applied Biosystems), 1.25 μl 1 μM forward-primer (A or B) and1.25 μl 1 μM reverse primer (C) or vice versa (Sigma Genosys), 5 μl DNA (10 ng/ul), 5 μl DNase, RNase free water to final volume 25 μl. Thermal profile of 7300 Real Time PCR System (Applied Biosystems) for allelespecific PCR: 50°C/2 min, 1×; 95°C/10 min, 1×; (95°C/0.15 min, 60°C/1 min)/50×; and dissociation of amplicons with incorporated SYBR green: 94°C/0.15 min; 60°C/0.30 min; increasing 1°C/min; 95°C/0.15 min. Amplification curves from real-time PCR with following dissociation curves revealed type A, or B of marker (M) allele (MA or MB). For each donor/recipient pair, there are different informative markers for relative quantification and chimerism estimation after allohSCT. Relative quantification was calculated and evaluated from amplification plots of the individual PCR reactions on 96-well plate by the gene expression Study software (7300 System SDS Software, Applied Biosystems). Amplification plot of each informative marker before alloTKB was used as the calibrator for relative quantification of measured marker and amplification plot of gene GADPH was used as endogenous control [40].

#### *3.2.3 Results*

In most of screened donor/recipient pairs, we have found 2–3 informative autologous and allogeneic markers. An example of donor and recipient (patient) screening for informative NPs DNA markers (M1–M12) by SYBR green-based real-time PCR amplification and following dissociation curves is presented in **Figure 5**. For this donor/recipient pair, recipient's marker 3B (M3B) can be considered as informative autologous marker and can be used for chimerism monitoring after allo-hSCT. Only in three cases, we did not find any of the informative autological DNA markers, which are most convenient (detection <1%) for the measurement of micro chimerism. After allo-hSCT, informative DNA markers found in samples of patient's peripheral blood of patients were quantified. The relative amounts (expressed in %) were evaluated from threshold cycle (cT) values of amplification curves and analysed by Study Software, previously used for a relative quantification of the gene expression. The results of the DNA marker quantification measurements were obtained from (relative quantification) RQ and simultaneously from the charts, which had been constructed automatically [41] and transformed (**Figure 6**). In this figure, chimerism monitoring of AML patient after allo-hSCT is expressed. All patients were regularly monitored, and from the quantification of autologous informative DNA markers, mixed (MC) or

*Monitoring of Chimerism in Rare Haematological Malignant Diseases after Allogeneic… DOI: http://dx.doi.org/10.5772/intechopen.89845*

#### **Figure 4.**

*Localisation of DNA markers (M1-M12) (11NPs).*

#### **Figure 5.**

*An example of screening DNA markers M1-M12 by SYBR green based real-time amplification and following dissociation curves. Recipient's marker 3B can be used for chimerism monitoring after allo-hSCT.*

complete (CC) chimerism was evaluated. Identification of donor allogenous informative markers have evaluated only semiquantitatively. Cutoff for CC was 0.01% with relative quantification sensitivity RQ = 0.0001. Special time-dependent charts for each patient were archived and sent to doctors in appropriate transplantation units.

#### *3.2.4 Conclusion*

We have implemented the method of DNA diagnostics for the detection of micro chimerism (add to cytogenetics and FISH previously used) after allo-hSCT, which is independent from the sex of donor and recipient and reaches sensitivity of <1% (0.01%). The method of relative quantification by real-time PCR is currently routinely used in our clinical practice and can serve clinician as another factor for the patients monitoring after haematopoietic stem cell transplantation and his early intervention if necessary.

#### **Figure 6.**

*Chimerism monitoring of AML patient after allo-hSCT by SYBR green based real-time PCR relative quantification method. MC, mixed chimerism; CC, complete chimerism.*

### **3.3 Different malignant and non-malignant diagnosis in our study and their donors provided by 'RQ PCR'**

#### *3.3.1 Results*

In our diagnostic laboratory, we have analysed samples from patients before and after allo-hSCT with different malignant and non-malignant diagnoses (**Figure 7**).

During the last decades, the number of allo-hSCT among *CML* patients is decreasing due to the drug-targeted treatment and transplantation is mostly indicated in the case of resistance to targeted tyrosine-kinase-inhibitor treatment. In our study, only 9 CML patients were transplanted from the cohort of 188 different malignant and non-malignant diagnosis (patients from University Hospital Bratislava, Slovak Republic), which is only 5%. The majority of allo-hSCT were provided and chimerism was monitored in AML patients (45%); then, ALL patients were 31% and MDS with others non-malignant diagnoses were 19% (11 + 8).

The selection of a donor is a critical element contributing to the success of haematopoietic cell transplantation. Possible donors for allo-hSCT can be HLA identical, haploidentical or mismatched (sibling, relative or unrelated donor). In our study, there were 188 donors (111 from relatives and 77 from Slovak National Bone Marrow Donor Registry and world registers of the bone marrow donors) and 188 recipients (**Table 1**). About 95% haemopoietic stem cells were obtained from peripheral blood by its drug mobilisation. Only 5% stem cells were from the bone marrow.

#### *3.3.2 Conclusion*

From our results, it is obvious that assessment of sex-independent donor/recipient method based on SYBR green real-time relative quantification RQ PCR) for chimerism monitoring has been very important due to cohort of different gender donor/recipient pairs. This molecular method has completed cytogenetics and FISH methods used previously for chimerism monitoring. The advantage of 'RQ PCR' compared to methods mentioned above is not only in its higher diagnostic and analytical sensitivity and sex-independent monitoring but also in the fact

*Monitoring of Chimerism in Rare Haematological Malignant Diseases after Allogeneic… DOI: http://dx.doi.org/10.5772/intechopen.89845*

#### **Figure 7.**

*Different malignant and non-malignant diagnosis in process of allo-hSCT*


#### **Table 1.**

*Types of recipient(patient)/donor variants in the process of allo-hSCT.*

that almost 50% of AML patients has not genetic mutation marker to distinguish increasing mix chimerism and no indicator for relapse of primary patient disorder before allo-hSCT.
