**2. Materials and methods**

#### **2.1 Study population**

250 subjects (144 men, 106 women) admitted to the Cellular Immunology and Cytometry and Cellular Therapy Laboratory of National Blood Transfusion Center of Tunis –Tunisia, for autologous PBPC mobilization were enrolled.

Our patients can be divided in 4 subgroups distributed as follows: Group 1: 85 Non-Hodgkin's Lymphoma (57 men, 28 women) which comprises 80 Diffuse B Cell Lymphoma, 4 Mantle Cell Lymphoma and a patient with Follicular Lymphoma.

Group 2: 87 Multiple Myeloma (48 men, 39 women).

Group 3: 63 Hodgkin's disease (31 men, 32 women).

Group 4: composed of 15 patients with Acute Myeloid Leukemia (9 men, 6 women).

Besides, a group composed of 41 subjects (24 men, 17 women) with mean age of 32 years (range 12-63 years) designated for peripheral blood stem cells (PBSC) mobilization. They were visiting the Cellular Immunology and Cytometry and Cellular therapy Laboratory of National Blood Transfusion Center of Tunis–Tunisia as allogenic donors for stem cell transplantation.

Then, a group of 165 healthy blood donors visiting the Blood Transfusion Service of National Blood Transfusion Center of Tunis -Tunisia served as a control group was enrolled in the study. Whole details concerning the subjects will be resumed in Table 1.

Written informed consent was obtained from all subjects according to a protocol approved by the ethical committee for scientific and medical research of the National Blood Transfusion Center and National Bone marrow transplantation center of Tunis (Tunisia) in accordance with the Declaration of Helsinki.

Circulating hematopoietic progenitors CD34+ were evaluated daily by flow cytometry and PBSC collections or apheresis were begun when peripheral CD34+ cells were ~20 cells/µl. Apheresis was usually performed daily using continuous flow blood cell separators COBE SPECTRA and MCS+.

#### **2.2 DNA extraction and genotyping**

Genomic DNA was prepared from EDTA anticoagulated peripheral blood by using a common salting-out procedure [Miller SA et al, 1988].

So, identifying SNPs predictive of poor or good response to G-CSF or any mobilization regimen, in terms of number of CD34+ cells mobilized, might be useful in discussing the possibility of using a different mobilizing agent or a different source of CD34+ cells for auto-

In this issue, we proposed to study the distribution of three genetic polymorphisms: SDF1- 3'A, MMP-9 C-1562T and GNB3 C825T in Tunisian patients with malignant hematological diseases who underwent stem cell mobilization for autologous transplantation compared to

250 subjects (144 men, 106 women) admitted to the Cellular Immunology and Cytometry and Cellular Therapy Laboratory of National Blood Transfusion Center of Tunis –Tunisia,

Our patients can be divided in 4 subgroups distributed as follows: Group 1: 85 Non-Hodgkin's Lymphoma (57 men, 28 women) which comprises 80 Diffuse B Cell Lymphoma,

Besides, a group composed of 41 subjects (24 men, 17 women) with mean age of 32 years (range 12-63 years) designated for peripheral blood stem cells (PBSC) mobilization. They were visiting the Cellular Immunology and Cytometry and Cellular therapy Laboratory of National Blood Transfusion Center of Tunis–Tunisia as allogenic donors for stem cell

Then, a group of 165 healthy blood donors visiting the Blood Transfusion Service of National Blood Transfusion Center of Tunis -Tunisia served as a control group was enrolled

Written informed consent was obtained from all subjects according to a protocol approved by the ethical committee for scientific and medical research of the National Blood Transfusion Center and National Bone marrow transplantation center of Tunis (Tunisia) in

Circulating hematopoietic progenitors CD34+ were evaluated daily by flow cytometry and PBSC collections or apheresis were begun when peripheral CD34+ cells were ~20 cells/µl. Apheresis was usually performed daily using continuous flow blood cell separators COBE

Genomic DNA was prepared from EDTA anticoagulated peripheral blood by using a

Group 4: composed of 15 patients with Acute Myeloid Leukemia (9 men, 6 women).

in the study. Whole details concerning the subjects will be resumed in Table 1.

HSCT and allo-HSCT.

a group of healthy allogenic PBPC donors.

for autologous PBPC mobilization were enrolled.

Group 2: 87 Multiple Myeloma (48 men, 39 women). Group 3: 63 Hodgkin's disease (31 men, 32 women).

accordance with the Declaration of Helsinki.

**2.2 DNA extraction and genotyping** 

common salting-out procedure [Miller SA et al, 1988].

4 Mantle Cell Lymphoma and a patient with Follicular Lymphoma.

**2. Materials and methods** 

**2.1 Study population** 

transplantation.

SPECTRA and MCS+.


Table 1. patients and healthy allogenic PBPC donors charachteristics Abbreviations: G-CSF, granulocyte colony-stimulating factor; G/C, G-CSF- chemotherapy; ICE, ifosfamide, carboplatin, etoposide; ESHAP/DHAP, etoposide, cytarabine, methylprednisolone,

Distribution of SDF1-3'A, GNB3 C825T and MMP-9 C-1562T Polymorphisms

in HSC CD34+ from Peripheral Blood of Patients with Hematological Malignancies 303

Fig. 2. MMP-9 genotyping by PCR-RFLP analysis followed by separation on 2% agarose gel as described in text. Lanes 1 and 8, 100 pb ladder; lanes 2 and 6, C/C; lanes 3 and 9, C/T;

Fig. 3. GNB3 C825T genotyping by PCR-RFLP analysis followed by separation on 2% agarose gel as described in text. Lane 1, 100 pb ladder; lanes 2 and 3, C/T; lane 4, T/T;

lane 5, T/T.

lane 5, C/C.

#### **2.3 Genotyping**

The reaction mixture consisted of 1µl PCR buffer 10x, 2 mM of MgSo4, 0.2 mM of each dNTP, 400mM of each primer, and 0,5units/reaction Taq DNA polymerase (Bio Basic Inc).

The reaction conditions were: For SDF1–3'A an initial denaturation at 95°C for five minutes, then 35 cycles at 94°C for 30 seconds, at 58°C for 30 seconds, at 72°C for 1min, and finally extension at 72°C for 7 minutes.

All specimens were examined for the presence of amplifiable DNA. PCR products were digested with 10units HpaII/reaction (Fermentas) at 37°C for overnight [Benboubker L et al, 2001] (figure 1).

For, MMP-9 C-1562T, PCR conditions as above, with annealing temperature at 67°C. PCR products were digested with 10units Hin1II/reaction (Fermentas) at 37°C for overnight [Zhang B et al, 1999; Toru Ogata et al, 2005] (figure 2).

For GNB3 C825T, the PCR-reaction began with denaturation at 95°C for 5 min, followed by 35 cycles of denaturation at 94°C (for 30 seconds), annealing at 55°C (30 s), extension at 72°C (1min), and a final extension at 72°C (7 min). PCR products were digested with BseDI at 60°C (4 h), separated on 2% agarose gels, and visualized under UV illumination [Cheng-Ho Tsai MD et al, 2000] (figure 3)

Fig. 1. SDF-1 genotyping by PCR-RFLP analysis followed by separation on 2% agarose gel as described in text. Lane 1, 100pb ladder; lanes 2 and 4, G/G; lanes 3 and 5, G/A; lane 11, A/A

The reaction mixture consisted of 1µl PCR buffer 10x, 2 mM of MgSo4, 0.2 mM of each dNTP, 400mM of each primer, and 0,5units/reaction Taq DNA polymerase (Bio Basic Inc). The reaction conditions were: For SDF1–3'A an initial denaturation at 95°C for five minutes, then 35 cycles at 94°C for 30 seconds, at 58°C for 30 seconds, at 72°C for 1min, and finally

All specimens were examined for the presence of amplifiable DNA. PCR products were digested with 10units HpaII/reaction (Fermentas) at 37°C for overnight [Benboubker L et al,

For, MMP-9 C-1562T, PCR conditions as above, with annealing temperature at 67°C. PCR products were digested with 10units Hin1II/reaction (Fermentas) at 37°C for overnight

For GNB3 C825T, the PCR-reaction began with denaturation at 95°C for 5 min, followed by 35 cycles of denaturation at 94°C (for 30 seconds), annealing at 55°C (30 s), extension at 72°C (1min), and a final extension at 72°C (7 min). PCR products were digested with BseDI at 60°C (4 h), separated on 2% agarose gels, and visualized under UV illumination [Cheng-Ho

Fig. 1. SDF-1 genotyping by PCR-RFLP analysis followed by separation on 2% agarose gel as described in text. Lane 1, 100pb ladder; lanes 2 and 4, G/G; lanes 3 and 5, G/A; lane 11, A/A

**2.3 Genotyping** 

2001] (figure 1).

extension at 72°C for 7 minutes.

Tsai MD et al, 2000] (figure 3)

[Zhang B et al, 1999; Toru Ogata et al, 2005] (figure 2).

Fig. 2. MMP-9 genotyping by PCR-RFLP analysis followed by separation on 2% agarose gel as described in text. Lanes 1 and 8, 100 pb ladder; lanes 2 and 6, C/C; lanes 3 and 9, C/T; lane 5, T/T.

Fig. 3. GNB3 C825T genotyping by PCR-RFLP analysis followed by separation on 2% agarose gel as described in text. Lane 1, 100 pb ladder; lanes 2 and 3, C/T; lane 4, T/T; lane 5, C/C.


Table 2. All genotyping Details, corresponding to each polymorphism studied are provided.

Distribution of SDF1-3'A, GNB3 C825T and MMP-9 C-1562T Polymorphisms

software (SPSS 16.0 for windows; SPSS Inc., Chicago, IL.).

**2.4 Statistical analysis** 

**3. Results** 

in HSC CD34+ from Peripheral Blood of Patients with Hematological Malignancies 305

Allele and genotype frequencies of the studied polymorphisms in patients and healthy controls were formulated by direct counting. Statistical analysis was performed using SPSS

The allele frequencies of SDF1-3'A, GNB3 C825T and MMP-9C-1562T polymorphisms were tested for the Hardy–Weinberg equilibrium of the whole group or subgroups of patients and were compared to the respective frequencies of the control group using the Pearson chi-square test or Fisher's exact test when appropriate. The same test was applied to compare the genotype frequency between patients and controls. Association of the allelic frequencies with the clinico-pathologic parameters was evaluated by χ2 test. The odds ratios (OR) and 95% confidence intervals (CI) were calculated too. P<0.05 was required for statistical significance.

**3.1 Patient's distributions according to their CD34+ cell yield and failure rates** 

cytotoxic chemotherapy than patients with MM [Iskra Pusic et al, 2008] (figure 4).

**to disease: A comparison between healthy donors of PBSC and patients** 

also for the CC and CT genotypes (p= 0.039; p= 0.004; Table 3).

and GA genotypes, respectively).

**3.2 Analysis of the studied polymorphisms in the 4 subgroups of patients according** 

According to this study, SDF1-3'A and MMP-9 C-1562 T polymorphisms were significantly different between the patients and healthy controls (table 3). Particularly, we found significant differences in all the allelic and genotypic frequencies of the SDF1-3'A polymorphism in the MM group (p<0.05; OR=3.245 CI (95%) [1.830-5.753] for A allele; p= 0.017; OR= 3.324 CI (95%) [1.182-9.348]; p= 0.009; OR= 2.072 CI (95%) [1.200-3.580] for AA

Concerning the MMP-9 C-1562 T polymorphism its distribution was significantly different in the same MM group of patients compared to the control group, significant differences were observed exclusively for the T allele (p=0.041; OR=2.295 CI (95%) [1.020-5.168]) and

Overall 83% of patients included in this study collected ≥2x106 CD34+ cells/kg after a maximum of 4 aphereses, among them 20% collected 2-5x106 CD34cells/kg, and 63% collected ≥5x106 CD34 cells/kg. Beside, 10% are remobilizers as they did not achieve the threshold of CD34+ cell yield of 2x106 CD34/kg within 4 apheresis days and are subjects to another mobilization protocol. Among them, the group of NHL represented the highest rate (40%), the lower ones, the group of MM and AML, which represented respectively 19% and 13%. By contrast, others are designed as first mobilizers (90%) since they have already collected ≥2x106CD34+ cells/kg after a maximum of 4 aphereses days. Amongst them the group of multiple myeloma was the most frequent (40%), thereafter the group of Non-Hodgkin's lymphoma (34%) and Hodgkin's disease with 26%. For the patients included in this study, mobilization failure was defined as <2x106 CD34+ cells/kg obtained within 4 apheresis days. So, especially MM patients collected ≥5x106CD34+ cells/kg and contained the highest CD34+ cell yield (8,89x106 CD34/kg for MM, and 5,51x106 CD34/kg for the others patients). Furthermore, the fact that MM patients had higher yield of CD34+ cells compared to NHL and HD is likely since that NHL and HL patients are frequently more heavily pretreated with

#### **2.4 Statistical analysis**

304 Advances in Hematopoietic Stem Cell Research

Table 2. All genotyping Details, corresponding to each polymorphism studied are provided.

Allele and genotype frequencies of the studied polymorphisms in patients and healthy controls were formulated by direct counting. Statistical analysis was performed using SPSS software (SPSS 16.0 for windows; SPSS Inc., Chicago, IL.).

The allele frequencies of SDF1-3'A, GNB3 C825T and MMP-9C-1562T polymorphisms were tested for the Hardy–Weinberg equilibrium of the whole group or subgroups of patients and were compared to the respective frequencies of the control group using the Pearson chi-square test or Fisher's exact test when appropriate. The same test was applied to compare the genotype frequency between patients and controls. Association of the allelic frequencies with the clinico-pathologic parameters was evaluated by χ2 test. The odds ratios (OR) and 95% confidence intervals (CI) were calculated too. P<0.05 was required for statistical significance.
