Journal of

Background and Aim: Acute promyelocytic leukemia (APL) is associated with the t(1517) ,fusing promyelocytic leukemia (PML) and retinoic acid receptor-a (RARa) genes. This disease is uniquely sensitive to treatment with all-trans retinoic acid (ATRA) and highly responsive to conventional chemotherapy. The t(1117)(q23q21) abnormality associated with a PLZF-RARa rearrangement is the commonest of the alternative translocations accounting for less than 1% APL. Blasts from PLZF-RARa cases have been found to be resistant to the differentiating effects of retinoids. In this study we aimed to determine the PLZF-RARa and fusion genes in patients with APL morphology who referred to Hematology-Oncology and BMT research center, Tehran Shariaty Hospital in 2006.

Materials & Methods: Peripheral blood and/or bone marrow samples were taken from 200 patients with APL morphology and 200 patients with other subtypes of AML. The mono-nuclear cells were enriched by centrifugation over a ficoll-isopaque gradient. RNA extracted by Trizol or TriReagent and then reverse transcribed to cDNA using random primers. PCR performed using specific primers for each fusion. PCR products electerophoresed on a 2% agarose gel containing 0.05% ethidiume bromide.

Results: In 2 (1%) patients with APL-morphology the RT-PCR analysis showed PLZF-RARa fusion transcripts.

Conclusion: It can be concluded that RT-PCR is a rapid and sensitive method for detection of abnormal fusion genes in leukemia and allows the definition of a correct strategy for treatment and subsequent minimal residual disease monitoring

Background and Aim: Chronic Myelogenous Leukemia (CML) is a clonal hematopoietic stem cell disorder characterized by a translocation between chromosome 9 and 22 called Philadelphia Chromosome. Telomerase- essential enzyme that adds telomeric repeats into the telomeres- maintains integrity of  chromosomal ends. Most normal somatic cells do not express hTERT (catalytic subunit of telomerase) but most neoplasia and cancer cells express it. In this study we evaluated the hTERT expression in patients with CML at different phases of the disease.

Materials and Methods: In this cross sectional study, 73 samples of 45 patients with CML were studied. Twenty six of samples were taken from patients in chronic phase before therapy and 26 samples three month after therapy. Nine samples were taken in accelerated phase and 12 in blastic phase. hTERT expression was studied by RT-PCR and the results were analyzed using SPSS 15.

Results: Thirty three (73%) of patients were male and 12 (23%) were female. Patients were divided into three age groups 17-29, 30-40 and 41-75 years. Of 73 samples, 43 samples (58.9%) were positive for hTERT and 30 samples (41.1%) were negative for this gene. In chronic phase (before therapy) 69.2% of the samples were PCR positive, but after therapy only 38.5% of samples were PCR positive. In accelerated and blastic phases, 55.6% and 83.3% of samples were PCR positive respectively. The hTERT positivity was differently significant (p<0.05) among different phases of the disease.

Conclusion: Significant difference between hTERT expression in different phases of CML disease can be used as a useful molecular marker for fallowing up, prognosis and disease progression after treatment

Background & Aim : Chronic myeloid leukemia (CML) is a disorder of pluripotential hematopoietic stem cell that is as a myeloproliferative disease and occurs in about 15 percent of all leukemia. Two cell cycle regulatory proteins that function as tumor suppressor are P16INK4A and P14ARF. The origin of these two proteins is a human INK4A-ARF gene locus that located on chromosome 9p21. P16INK4A control retinoblastoma (Rb) and P14ARF control with p53 thought negative feedback. The purposes of this study, this was that whether these genes are preferable use as a factor in prognosis and progression of disease.

Materials and Methods: This research was a Cross sectional study.  The expression of p16INK4A and p14ARF mRNA in about 73 peripheral bloods (PB) Samples were collected from 45 CML patients at different phases of disease were assayed by reverse transcriptase polymerase chain reaction (RT-PCR). 26 samples were from patients at chronic phase before any treatment, 26 samples 3 month after treatment with imatinib, 9 samples in accelerated phase and 12 samples in Blastic phase.

Results. From 45 patients with CML, 33 patients (73%) were men and 12 patients (27%) were women. About 26 samples (35%) were p16INK4A positive and 55 samples (75%) were p14ARF RT-PCR positive. This expression of the two genes at different phases of disease were not statistically significant (p>0.05).

Conclusion: High percentage of the CML patients expressed P14ARF and P16INK4A genes. The expression of these gene at different phases of disease (diagnosis, accelerate, and Blastic phases) was not statistically significant even though, the expression of these genes was higher after the treatment.  The increased expression of these genes was probably because of the Imatinib treatment.

Background and Aim : Myeloproliferative neoplasms are clonal and heterogeneous disorders of hematopoietic stem cells lead to increase of one or more cell lines in the blood. Recently, the acquired mutation JAK2 V617F has been described in the majority of patients with myeloproliferative neoplasms (MPNs).This mutation is characterized by a G to T transverse at nucleotide 1849 in exon 12 of the JAK2 gene, located on the chromosome 9p, leading to a substitution of valine to phenylalanine at amino acid position 617 in the JAK2 protein. The aim of this study was to assess the prevalence of JAK2 mutation in MPN patients.

Materials and Methods: In this experimental study we evaluated JAK2 mutation in 58 patients with MPNs by simple randomized sampling. The mutation was detected by ARMS-PCR in patients.

Results: The JAK2 V617F mutation was detected in 86.6% (26/30) of patients with polycythemia Vera, 46.6% (7/15) of patients with essential thrombocythemia and 61.5% (8/13) of patients with idiopathic myelofibrosis. Polycythemia Vera patients carrying the mutation displayed a higher levels of WBC (p=0.03) and 61.5% (16/26) of these patients were females. The differences in other groups were not significant. The mutation was confirmed by sequencing.

Conclusions: Our Results show similarity with other studies. Thus, ARMS-PCR can be applied as differential diagnosis test for detection of JAK2 mutation in suspected patients with MPNs.

Background and Aim: The JAK2 is an acquired mutation that is observed in majority of patients with classical Philadelphia-negative Myeloproliferative neoplasms that include polycythemia vera (PV), essential thrombocythemia (ET), primary myelofibrosis (PMF). This acquired mutation is characterized by a G to T transversion at nucleotide 1849 in exon 12 of the JAK2 gene, leading to a substitution of valine to phenylalanine at amino acid position 617(V617F) of the JAK2 protein, and result in constitutive JAK2 activation that promotes hypersensitivity to growth factors and cytokines.

Materials and Methods: In this study we evaluated RNA from 58 patients with MPNs and statistical analysis was done with mann whitney test. The mutation detected by AS-PCR. In addition, 3 samples were sequenced in Mille gen company.

Results: 46 patients:86.6%(26/30) of those with polycythemia vera, 53.3% (8/15) of those with essential thrombocythemia,61.5% (8/13) of those with idiopathic myelofibrosis polycythemia vera patient carrying the mutation displayed higher levels of WBC (p=0.03). on the other hand,16 out of 26 JAK2V617F positive patients were female there is a demonstrate correlation between the presence of a mutant allele and female gender. The difference in other groups were not significant.

Discussion and Conclusion: The JAK2V617F mutation has been detected in the vast majority of patients with polycythmia vera (65-95%) and in a lower frequency in patients with essential thrombocythemia (23-57%), idiopathic myelofibrosis (23-57%) and chronic myeloid leukemia 19% (3/16 CML Ph-). Detection of the mutation is helpful in differential diagnosis, prognosis, and prediction of therapeutic response.

Background and Aim: APL is a Prevalent leukemia that Approximately included 5-10% of patients with acute myeloblastic leukemia. ATRA and recently arsenic is used for treatment. ATRA leadsto resistance to treatment and arsenic is toxic in high doses.AZT induce cell death in different ways. The purpose of this study was Assessment of effect of AZT, a telomerase inhibitor, on NB4 cell line (APL cell line) to reduce toxic effect of high dose arsenic.

Materials and Methods: In this study, viability and metabolic activityof NB4 cells, treated by different concentrations of AZT(50,100,200 µM), was assessed by trypan blue dye method and MTT assay respectively.

Results: Treated cells with AZT=50,100,200µM showed decreased viability, both in dose-dependent and time-dependent through trypan blue dye method and decreased cell metabolic activity by MTT assay.

Discussion and Conclusion: Considering that AZT is able to induce apoptosis and decrease cell activity, it seems AZT is a suitable drug for inhibiting the growth of tumor cells.

Background and Aim: Acute promyelocytic leukemia (APL) is a distinct subtype of acute myeloid leukemia. APL is characterized by a balanced reciprocal translocation between chromosomes 15 and 17, t(517)). Important therapeutic strategies for this disease are ATRA and Arsenic trioxide. To eliminate tumor cells with arsenic, high dose of arsenic is needed. But high dose is toxic for normal tissue. The purpose of this study is Assessment of effect of low dose of arsenic trioxide in combination with AZT on NB4 cell line (APL cell line) to reduce toxic effect of high dose arsenic.

Materials and Methods: In this study after NB4 cell line culture and proliferation, the cells treated with low dose of arsenic trioxide(0.5µM) in combination with different doses of AZT(50, 100, 200 µM) and then viability and metabolic activity was assessed by try pan blue and MTT assay respectively.

Results: Low dose of arsenic (0.5µm) alone and in combination with dose of 50µm of AZT has little effect on viability and metabolic activity but in combination with higher dose of AZT has significant effect on viability and metabolic activity and both viability and metabolic activity significantly reduced.

Conclusion: Different apoptosis- induced mechanisms cause apoptosis by arsenic and AZT. Since some of these mechanisms between AZT and arsenic are similar, so maybe these similar mechanisms cause synergic effect and significant reduction of viability and metabolic activity in combination of these two drugs.

Background and Aim: Tumor dissemination via blood to distant organs is the main cause of death. Therefore, there is a critical need to set up sensitive methods for the early detection of circulating tumor cells(CTCs) and disseminated tumor cells(DTCs) in peripheral blood (PB) and bone marrow(BM) specimens of breast cancer patients. The aim of this research is to study the detection of micrometastasis using MUC2 in such patients.

Materials and Methods: In this study, PB and BM samples were collected from 50 breast cancer patients after operation and before adjuvant therapy. Mucin 2 (MUC2) was used as a tumor marker and its transcript level in the sample patients was analyzed using gene specific, quantitative real-time PCR reaction with SYBR Green technology. Samples from 20 healthy individuals were used as negative controls. HPRT was used as a reference gene.

Results: MUC2 mRNA was detected in 8 (16%) of PB and BM samples. MUC2 mRNA was not detected in PB samples of healthy individuals. The relapse rate among MUC2-positive patients was higher than MUC2-negative patients and it was statistically significant in BM (P<0.05).

Conclusion: This study shows that MUC2 can be a suitable marker for the detection of micrometastasis in breast cancer patients at early stages of cancer and that it may provide the basis for identifying women at risk of relapse.

 Background and Aim: A goal of modern cancer research is to reach targeted therapies with drugs having fewer side effects. AZD1152 is a highly specific inhibitor of Aurora Kinase B, which leads to the programmed cell death by different mechanisms. The aim of this study was to evaluate the effects of AZD1152 on viability and metabolic activity of NB4 cells (APL derived cell line).

 Materials and Methods: The cells were treated with various concentrations of AZD1152. After 24, 48 and 72h treatments, the metabolic activity and viability of inhibitor-treated NB4 cells were assessed using MTT and trypan blue dye exclusion assays, respectively. Data were analyzed by applying student’s t-test (Microsoft Excel).

 Results: A t 25, 50 and 100 nM, AZD1152 reduced the metabolic activity by 9.2, 15.5 and 56.2% (after 24h), 10.3, 19.5 and 59.9% (after 48h), and 17.1, 28.4 and 64.8% (after 72h), respectively. Meanwhile, the percentage of viability was decreased to about 51, 45 and 40% (after 24h), 39, 36 and 30% (after 48h), and 34, 32 and 28% (after 72h), respectively.

 Conclusion : According to the results, AZD1152 has substantial efficacy on APL cell line and may be applied in some cases, e. g., for patients who have relapse or who become refractory to the conventional chemotherapy. Further studies are needed to show the molecular mechanisms regulating effects of this anti-cancer agent.

Background and Aim: Acute promyelocytic leukemia (APL) is a distinct type of leukemia which is caused due to a blockage in myeloid cells normal maturation. The most important therapeutic strategies include the use of ATRA and Arsenic trioxide. Although ATRA is generally well tolerated, some patients develop Retinoic acid syndrome. Some of the symptoms of this syndrome are directly or indirectly related to elevated WBC counts. This study aims to determine the effect of ATRA and BIBR1532 combination on WBC count as a factor leading to the formation of ATRA syndrome.

Materials and Methods: To investigate the effect of BIBR1532 and ATRA combination, NB4 cells were cultured in the presence of 30μ M and 1 μM densities of the drugs. To study the effect of drugs on living cells count, proliferation activity, and metabolic activity of the cells, Trypan blue, Brdu and MTT tests were used, respectively.

Results: The results of Trypan blue, MTT and Brdu suggest that the combination of ATRA and BIBR1532 is more effective than ATRA alone on the reduction of viable cell count, metabolic activity and proliferation of leukemic cells in the first five days of treatment.

Conclusion: The results suggest that the combination of ATRA and BIBR1532 is probably more effective in the treatment of APL patients. It seems that such improvement in results is more obvious especially among the patients who are at a higher risk of ATRA syndrome.