marjan yaghmaie

Despite the advances in treatment, breast cancer (BC) remains a major cause of death in women. Thisstudy aims to evaluate the prognostic significance of detecting circulating tumor cells (CTCs) and disseminated tumorcells (DTCs) in paired peripheral blood (PB) and bone marrow (BM) samples obtained both before and after adjuvantchemotherapy from patients with operable BC. In this experimental study, from 160 patients with primary BC, we collected 160 PB and BM samplesbefore and we could be able to collect PB and BM samples from 100 of them after adjuvant chemotherapy. The expressionlevel of cytokeratin 19 (CK19), carcinoembryonic antigen (CEA), mammaglobin 1 (MGB1), mucin 2 (MUC2) and trefoil factor1 (TFF1) mRNAs in the PB/BM samples were analyzed by quantitative real-time polymerase chain reaction (PCR). Multivariate Cox regression analyses indicated that the detection of CK19 mRNA-positive CTCs/DTCs eitherbefore or after adjuvant chemotherapy was an independent factor for prognosis associated with decreased diseasefreesurvival (DFS). Patients with tumor cells detected in both PB and BM and patients with persistent detection oftumor cells before and after chemotherapy had worse outcomes compared to those with tumor cells detected in one orneither of the compartments. This study suggests that the detection of CK19 mRNA-positive CTCs/DTCs either before or after adjuvantchemotherapy could be an independent predictor of DFS in operable BC patients.

Placenta-specific 1 (PLAC1) is one of the cancer-testis-placenta antigens that has no expression in normal tissue except placenta trophoblast and testicular germ cells, but is overexpressed in a variety of solid tumors. There is a lack of studies on the expression of PLAC1 in leukemia. We investigated expression of PLAC1 in Acute Myeloid Leukemia (AML) and Acute Lymphoblastic Leukemia (ALL).

In this study, we investigated expression pattern of PLAC1 gene in peripheral blood and bone marrow mononuclear cells of newly-diagnosed patients with AML (n=31) and ALL (n=31) using quantitative real-time PCR. Normal subjects (n=17) were considered as control. The PLAC1 protein expression in the samples were also detected using western blotting.

Our data demonstrated that PLAC1 transcripts had 2.7 and 2.9 fold-change increase in AML and ALL, respectively, compared to normal samples. PLAC1 transcript expression was totally negative in all studied normal subjects. Level of PLAC1 mRNA expression in ALL statistically increased compared to normal samples (p=0.038). However, relative mRNA expression of PLAC1 in AML was not significant in comparison to normal subjects (p=0.848). Furthermore, relative mRNA expression of PLAC1 in AML subtypes was not statistically significant (p=0.756). PLAC1 gene expression showed no difference in demographical clinical and para-clinical parameters. Western blotting confirmed expression of PLAC1 in the ALL and AML samples.

Considering PLAC1 expression profile in acute leukemia, PLAC1 could be a potential marker in leukemia which needs complementary studies in the future.

Although Imatinib has revolutionized the treatment of chronic myeloid leukemia (CML), not all patients reach complete remission and a considerable proportion of the patients develop resistance to Imatinib.

In an attempt to increase the tail on the survival curve, we conducted a Phase I/II study of PR1/BCR-ABL multipeptides vaccination trial in CML patients with at least 15 months of Imatinib treatment and 5 months of persistent molecular residual disease.

One month after the completion of the vaccinations, 4 patients nearly developed a 1-log fall in their BCR-ABL transcript level, with 4 patients achieving a major molecular response (MMR). Nine patients were followed for more than a period of 7 years. The vaccinations were associated with a MMR in five patients and a complete molecular response (CMR) in one patient. The removal of Imatinib in two patients who achieved MMR after the vaccinations led to a resurgence of the leukemia population and relapse.

Our study suggests that a combination of immunotherapy with Imatinib targeted therapy keeps the leukemia population under control, improving the long-lasting clinical and molecular response of CML patients, for at least 7 years.

Current treatment options of acute lymphoblastic leukemia(ALL) include chemotherapy alone or hematopoietic stem cell transplantation (HSCT) following induction chemotherapy both along with CNS prophylaxis. The usual and standard induction regimens currently administered could have severe complications and mortality.

To lessen induction regimen complications in ALL patients who undergo HSCT, we used a cytoreduction induction regimen including dexamethasone (8 mg, IV, three times a day, for 28 days) and vincristine(1.4 mg/m2, IV, on days 1,8,15 and 22) for 49 newly diagnosed adult ALL patients followed by an early sibling donor HSCT within two months. The results were matched with outcomes of HSCT in 172 ALL patients inducted by standard induction regimen.

Median follow-up time was 5.41 years in the standard group and 5.27 years in the other. All patients of the case group (100%) achieved complete remission. Landmark analyses were performed to scrutinize the effect of treatments on different time intervals: first two years and 2nd to end years. Type of treatment had no significant effect on the hazard of death in the first landmark (HR=0.87, P=0.64). Cytoreduction regimen amplified the hazard of death 3.43 times more than the standard regimen in the second landmark (HR=3.43 P=0.035). Multivariate analysis showed that the cytoreduction regimen reduced the hazard of relapse about 22%, but not statistically significant (HR=0.78, P-value=0.24).

Overall, it seems despite achieving complete remission in induction therapy, depth of response is a critical predictor for long-term outcomes of HSCT in ALL patients, and the use of multiple agents may be necessary to decrease tumor cell burden and minimal residual disease(MRD).

Placenta-specific protein 1 (PLAC1) is one of the members of cancer-testis antigens family that has limited expression in normal tissue, but is upregulated in a variety of malignant tissues. Considering the lack of studies on the expression of FCRL1 in CML and CLL, the current study was conducted to examine the expression pattern of PLAC1 gene in these leukemias.

Fresh peripheral blood samples were collected from 6 CML and 10 CLL patients. In addition, peripheral blood samples of 10 healthy individuals were collected in EDTA as control group. All patients and healthy individuals signed a consent letter before sampling. The mononuclear cells were separated using ficoll-hypaque gradient centrifugation. Isolated mononuclear cells were used for RNA extraction and cDNA synthesis using RT-PCR method. Then, PLAC1 transcript expression in comparison to GAPDH were detected via Real-Time PCR. The statistical analyses were performed using chi-square test in SPSS.

All 10 normal samples were negative for PLAC1. The PLAC1 expression was found to be statistically different in CML group (4 out of 6 cases) compared with that in the normal group (P value = 0.000). However, CLL revealed no significant difference compared to normal individuals for PLAC1 expression (P Value = 0.648). In a significant percentage of CML patients, PLAC1 expression was positive but in CLL patients PLAC1, transcript expression was not evident.

It seems that PLAC1 could potentially be proposed as a biomarker in CML to aid in the diagnosis, prognosis, and treatment in the future.

Thyroid cancer is one of the most common endocrine malignancies. Anaplastic thyroid cancer is a rare and dead full cancer among types of the thyroid cancer. Despite the conventional chemotherapy, a considerable number of the patients show developing chemo resistance. Therefore, there is a necessary need to find the novel therapeutic approaches in the anaplastic thyroid cancer patients. The aim of this study was to study anti-tumor effect of Abemaciclib on the anaplastic thyroid carcinoma cell lines.

The human anaplastic thyroid cancer (SW1736 and C643) were cultured according to ATCC recommendations. The MTT assay was used to assess the chemo sensitivity of the cell lines in exposure to the desire concentration of Abemaciclib. Colony formation assay was used to determine the ability of the cell line colony formation in exposure to the drug. Quantitative real-time PCR was applied to analyze the mRNA expression of the apoptotic and anti-apoptotic genes.

The cell viability and proliferation of the thyroid cancer cell lines were remarkably inhibited in doses of 10 and 20 μM of Abemaciclib (p<0.0001). Also, Abemaciclib reduced the number of the SW1736 and C643 colonies in doses of 1 and 2.5 μM, respectively. Moreover, Abemaciclib significantly reduced anti-apoptotic gene expression levels, including BCL2 and CMYC, and increased pro-apoptotic gene expression levels, including p21 and BAX (p<0.05).

Our study suggests that Abemaciclib could be used as a therapeutic agent in anaplastic thyroid cancer. Further laboratory and animal studies are needed and recommended to evaluate the exact molecular and clinical properties of Abemaciclib.

Thyroid cancer is the most prevalent endocrine malignancies globally. Anaplastic thyroid carcinoma (ATC) accounts for 1-3% of all Thyroid cancer. The evidence showed that ATC is a highly invasive solid tumor with poor prognosis. Despite conventional chemotherapy treatments, a considerable number of patients show developing resistance to therapeutic agents and tumor relapse. The aim of this study was the investigation anti-tumor effect of Abemaciclib (novel targeted cancer therapy drug) on Anaplastic Thyroid carcinoma SW1736 and C643 cell lines.

SW1736 and C643 cell lines were treated by desire concentrations of Abemaciclib (0, 1, 2.5, 5, 10, and 20 μM) and cell viability was measured by MTT assay. Also, Anoikis resistance assay was conducted for non-adherent the cells in the exposure of Abemaciclib. The gene expression of apoptotic and anti-apoptotic genes was conducted by quantitative Real-time PCR.

Abemaciclib at the concentration of 10 and 20 μM effectively reduced cell proliferation and growth of the ATC cells compared to the control (p=0.000). Furthermore, we showed that 10 and 20 μM doses of the Abemaciclib inhibited the non-adherent ATC cells which were resistant to Anoikis death significantly (p=0.001). Moreover, we demonstrated this targeted therapy significantly reduced anti-apoptotic gene expression levels (BCL2 and CMYC) (p<0.05) and increased apoptotic gene expressions such as P21 and BAX (p<0.05).

Our data suggested that Abemaciclib can be utilized as a novel therapeutic agent in ATC cancer. Further in vivo and in vitro investigations are needed to evaluate molecular and clinical mechanisms of Abemaciclib.

Most of Gastric Cancer (GC) patients are diagnosed at an advanced stage with poor prognosis. Hypermethylations of several tumor suppressor genes in cell-free DNA of GC patients have been previously reported. In this study, an attempt was made to investigate the methylation status of P16, RASSF1A, RPRM, and RUNX3 and their potentials for early diagnosis of GC.

Methylation status of the four tumor suppressor genes in 96 plasma samples from histopathologically confirmed gastric adenocarcinoma patients (Stage I-IV) and 88 healthy controls was determined using methylation-specific PCR method. Receiver operating characteristic curve analysis was performed and Area Under the Curve (AUC) was calculated. Two tailed p<0.05 were considered statistically significant.

Methylated P16, RASSF1A, RPRM, and RUNX3 were significantly higher in the GC patients (41.7, 33.3, 66.7, and 58.3%) compared to the controls (15.9, 0.0, 6.8, and 4.5%), respectively (p<0.001). Stratification of patients showed that RPRM (AUC: 0.70, Sensitivity: 0.47, Specificity: 0.93, and p<0.001) and RUNX3 (AUC: 0.77, Sensitivity: 0.59, Specificity: 0.95, and p<0.001) had the highest performances in detection of early-stage (I+II) GC. The combined methylation of RPRM and RUNX3 in detection of early-stage GC had a higher AUC of 0.88 (SE=0.042; 95% CI:0.793–0.957; p<0.001), higher sensitivity of 0.82 and reduced specificity of 0.89.

Methylation analysis of RPRM and RUNX3 in circulating cell free-DNA of plasma could be suggested as a potential biomarker for detection of GC in early-stages.

Gastric cancer (GC) is considered as one of the most common types of cancer worldwide with poor prognosis and generally limited treatment options. Recent studies have indicated that HER2, MDM2, MYC, MET, and TP53 play an important role in the development of gastric cancer. Therefore, the aim of this study was to evaluate the incidence of amplification/deletion of these genes in patients with gastric cancer.

In this descriptive study, a total of 37 gastric cancer tissue samples from GC patients including 23 males (62.2%) and 14 females (37.8%) referred to the Hematology-Oncology and Stem Cell Research Center of Shariati Hospital, Tehran, from March 2015 to February 2016 were evaluated. The patient's age at diagnosis ranged from 33 to 85 years (median: 65 years). The amplification pattern of HER2, MDM2, MYC and MET genes and TP53 deletion were investigated by fluorescence in situ hybridization (FISH) technique performed on 3 to 5 micron section obtained from formalin-fixed and paraffin-embedded cancer tissues.

The tumors were preferably identified at the distal stomach (54.05%) in comparison to tumors arising from the gastric cardia. The tumor size varied between 2 and 5 cm (average, 3.5 cm). Seven of the cases (19%) had advanced tumors at the time of diagnosis. HER2, MDM2, MYC, MET and TP53 copy number alteration were successfully determined in all samples obtained from the GC patients. HER2, MDM2, and c-MYC genes were amplified in 2 (5.41%), 1 (2.7%) and 3 (8.11%) of 37 patient samples, however, MET gene amplification and TP53 deletion were not observed in the obtained GC tissue samples. Co-amplification of HER2, MDM2, and MYC genes, and co-amplification of HER2 and MYC genes were detected in one patient.

The results of this study indicate the low frequency of MDM2, HER2 and MYC genes in gastric cancer patient and their copy number alterations may provide diagnostic and prognostic marker for GC patients.

Zinc-finger Enhancer Binding protein (ZEB1) acts as a transcription factor to promote cancer progression through regulating Epithelial to Mesenchymal Transition (EMT). It is well-known that ZEB1 mRNA expression is directly induced by both Estrogen Receptor (ER) and Progesterone Receptor (PR). Moreover, Androgen Receptor (AR) and PR could bind to the same regulatory element. Since it has been shown that AR overexpresses in Gastric Cancer (GC) as a male-predominant tumor, the goal of this study was to evaluate whether AR could regulate ZEB1 expression in GC.

The expression profile of ZEB1 in 60 fresh GC and adjacent non-tumor tissues and 50 normal gastric specimens was assessed by qRT-PCR, and the association of ZEB1 expression with clinicopathological features was investigated. Furthermore, possible correlation between ZEB1 and AR was evaluated to elucidate a novel prognostic marker using Kaplan-Meier method and Cox regression model. Finally, molecular interaction of ZEB1 and AR was assessed using a potent AR antagonist in GC cells.

Among GC patients, 70.2% (40/57) overexpressed ZEB1 and 64.91% (37/57) overexpressed AR relative to normal gastric tissues. ZEB1 overexpression was significantly correlated with the AR overexpression in GC patients. Moreover, ZEB1 overexpression was remarkably associated with lower overall survival; however, it was not an independent prognostic factor. Evidence shows that simultaneous evaluation of ZEB1 and AR expression could independently predict survival of GC patients (HR= 2.193, p=0.047).

These findings have clinical importance suggesting simultaneous evaluation of ZEB1 and AR expression as a potential prognostic marker. Moreover, AR may regulate ZEB1 expression in GC cells proposing a possible promising targeted therapy for GC patients.

The analysis of the gene copy number alterations in tumor samples are increasingly used for diagnostic and prognostic purposes in patients with gastric cancer (GC). However, these procedures are not always applicable due to their invasive nature. In this study, we have analyzed the copy number alterations of five genes (HER2, MDM2, c-MYC, c-MET, and TP53) with a fixed relevance for GC in the circulating tumor cells (CTCs) of GC patients, and, accordingly, as a potential approach, evaluated their usage to complete primary tumor biopsy.

We analyzed the status of the copy number alterations of the selected genes in CTCs and matched biopsy tissues from 37 GC patients using fluorescence in situ hybridization.

HER2 amplification was observed in 2 (5.41%) samples. HER2 gene status in CTCs showed a strong agreement with its status in 36 out of 37 patients’ matched tissue samples (correlation: 97.29%; Kappa: 0.65; p < 0.001). MDM2 amplification was found only in 1 (2.70%) sample; however, the amplification of this gene was not detectable in the CTCs isolated from this patient. c-MYC amplification was observed in 3 (8.11%) samples, and the status of its amplification in the CTCs indicated a complete agreement with its status in the matched tissue samples (correlation: 100%; Kappa: 1.0).

Our work suggests that the amplification of HER2 and c-MYC is in concordance with the CTCs and achieved biopsies, and, consequently, CTC may act as a non-invasive alternative for recording the amplification of these genes among GC patients.

It is well-known that Aurora kinase A (AURKA) shows oncogenic properties in various tumor types including gastric cancer (GC). Moreover, previous studies have demonstrated that AURKA has a specific androgen receptor (AR) binding site in its promoter; thus, it could be regulated by AR. Since it has been shown that AR overexpresses in gastric cancer (GC) as a male-predominant tumor, the goal of this study was to evaluate the association between AR and AURKA and its prognostic value in GC patients.

We assessed the expression profile of AURKA in 60 fresh GC and adjacent non-tumor tissues and 50 normal gastric specimen by qRT-PCR, and investigated the association of AURKA expression with clinicopathological features. Furthermore, we evaluated possible correlation between AURKA and AR to elucidate a novel prognostic marker using Kaplan-Meier method and Cox regression model.

Among GC patients, 65% (39/60) overexpressed AURKA relative to normal gastric tissues. AURKA overexpression was significantly correlated with the AR overexpression in GC patients. Although AURKA expression alone was not remarkably associated with poor outcome, we provided some evidence that combined evaluation of AURKA and AR expression could independently predict survival of GC patients adjusted for other variables (HR=1.7, CI=1.314-3.833 p=0.042).

These results indicate that AR and AURKA may crosstalk to promote GC progression. Our findings have clinical importance because they suggest simultaneous assessment of AURKA and AR expression as a novel potential prognostic marker.

Myeloid cell leukemia-1 (Mcl-1) plays a pivotal role in the survival of hematologic and solid tumors, and is known as a substantial oncogene. Studies have demonstrated the altered expression of Mcl-1
has been linked to malignancy development and poor prognosis. In this research, we have studied the expression of Mcl-1 mRNA in myelodysplastic syndrome (MDS) patients and determined association with clinico-pathological factors, MDS subgroups as well as international prognostic scoring system. The relative level of Mcl-1 was determined by real time quantitative real-time polymerase chain reaction and gene expression normalized to Glyceraldehyde-3-phosphate dehydrogenase. Results indicated amplification of mRNA encoding Mcl-1 in 100% of the cases. The higher level of Mcl-1 existed in MDS patients compared with the healthy controls but there was no statistically difference of Mcl-1 expression between these groups. Fold change in gene expression was higher in advanced stage MDS, high risk MDS, cases with >5% blast and LDH >400 to their corresponding groups. In addition, the correlation between gene expression and cytogenetic prognostic subgroups was statistically significant (p=0.043). In the present study, we showed that Mcl-1 is expressed in MDS independent of the World Health Organization subgroup and international prognostic scoring system. Therefore, Mcl-1 may be up-regulated already in early stages of leukemogenesis.

Chromosomal translocations are among the most common mutational events in cancer development, especially in hematologic malignancies. However, the precise molecular mechanism of these events is still not clear. It has been recently shown that alternative non-homologous end-joining (alt-NHEJ), a newly described pathway for double-stranded DNA break repair, mediates the formation of chromosomal translocations. Here, we examined the expression levels of the main components of alt-NHEJ (PARP1 and LIG3) in acute myeloid leukemia (AML) patients and assessed their potential correlation
with the formation of chromosomal translocations.
This experimental study used reverse transcription-quantitative polymerase chain reaction (RTqPCR) to quantify the expression levels of PARP1 and LIG3 at the transcript level in AML patients (n=78) and healthy individuals (n=19).
PARP1 was the only gene overexpressed in the AML group when compared with healthy individuals (P=0.0004), especially in the poor prognosis sub-group. Both genes were, however, found to be up-regulated in AML patients with chromosomal translocations (P=0.04 and 0.0004 respectively). Moreover, patients with one isolated translocation showed an over-expression of only LIG3 (P=0.005), whereas those with two or more translocations over-expressed both LIG3 (P=0.002) and PARP1 (P=0.02).
The significant correlations observed between PARP1 and LIG3 expression and the rate of chromosomal translocations in AML patients provides a molecular context for further studies to investigate the causality of this association.

Soft tissue sarcomas (STS) constitute an uncommon and heterogeneous group of tumors of mesenchymal origin and various cytogenetic abnormalities ranging from distinct genomic rearrangements, such as chromosomal translocations and amplifications, to more intricate rearrangements involving multiple chromosomes. Fluorescence in situ hybridization (FISH) can be used to identify these chromosomal translocations and amplifications, and sub classify STS precisely. The current study aimed at investigating the usefulness of FISH, as a diagnostic ancillary aid, to detect cytogenetic abnormalities such as MDM2 (murine double minute 2) amplification and CHOP(C/EBP homologous protein) rearrangement in liposarcoma, as well as SYT (synaptotagmin) rearrangement in synovial sarcoma.
The FISH technique was used to analyze 17 specimens of liposarcoma for MDM2 amplification and CHOP rearrangement, and 10 specimens of synovial sarcoma for SYT rearrangement. The subtypes of liposarcoma and synovial sarcomas were reclassified according to the FISH results and compared with those of the respective histological findings.
According to the FISH results in 17 liposarcoma cases, well-differentiated liposarcoma(WDLPS), dedifferentiated liposarcoma (DDLPS), and myxoidliposarcoma (MLPS)subtypes were 41%, 53%, and 6%, respectively. In different subtypes of liposarcoma, a total of 30% mismatches were observed between pathologic and cytogenetic results. According to the histological findings from FISH analysis, SYT rearrangement was found only in three out of 10 (30%) synovial sarcomas.
The detection of cytogenetic abnormalities in patients with liposarcoma and synovial sarcoma by FISH technique provides an important objective tool to confirm sarcoma diagnosis and sub classification of specific sarcoma subtypes in such patients.

Myelodysplastic syndromes (MDSs) include a diverse group of clonal bone marrow disorders characterized by ineffective hematopoiesis and pancytopenia.It was found that down regulation of APAF1, a putative tumor suppressor gene (TSG),leads to resistance to chemotherapy and disease development in some cancers. In this study, we investigated the relation of APAF1 methylation status with its expression and clinicopathological factors in myelodysplastic syndrome (MDS) patients.
Methylation Sensitive-High Resolution Melting Curve Analysis (MS-HRM) was employed in studying the methylation of CpG islands in the APAF1promoter region in MDS. Gene expression was analyzed by using real time RT-PCR.
42.6% of patient samples were methylated in promoter region of APAF1 analyzed, while methylation of the genewas not seen in controls (P Our study indicated that APAF1hypermethylation in MDS was associated to high-risk disease classified according to the IPSS, WHO and cytogenetic risk.

Acute promyelocytic leukemia (APL) is a unique subtype of acute leukemia. APL is a curable disease; however, drug resistance, early mortality, disease relapse and treatment-related complications remain challenges in APL patient management. One issue underlying these challenges is that the molecular mechanisms of the disease are not sufficiently understood.
In this study, we performed a meta-analysis of gene expression profiles derived from microarray experiments and explored the background of disease by functional and pathway analysis.
Our analysis revealed a gene signature with 406 genes that are up or down-regulated in APL. The pathway analysis determined that MAPK pathway and its involved elements such as JUN gene and AP-1 play important roles in APL pathogenesis along with insulin-like growth factor–binding protein-7.
The results of this meta-analysis could be useful for developing more effective therapy strategies and new targets for diagnosis and drugs.

BackgroundAtaxia telangiectasia (A-T) is a common genetically inherited cause of early childhood-onset ataxia. The infrequency of this disease, vast phenotype variation, disorders with features similar to those of A-T, and lack of definite laboratory test, make diagnosis difficult. In addition, there is no rapid reliable laboratory method for identifying A-T heterozygotes, who susceptible to ionizing radiation (IR), atherosclerosis, diabetes, and cancers. We used SMC1pSer966 (pSMC1) in-cell colorimetric ELISA to diagnosis and screen in A-T families.
With informed consent, 2cc peripheral blood was collected from the 15 A-T patients, their parents, and 24 healthy controls with no family history of malignancy, diabetes, and atherosclerosis. Extracted peripheral blood mononuclear cells (PBMCs) were cultured in poly-L-Lysine treated 96-well plate with density of 70,000 cells per well. SMC1 phosphorylation was evaluated with cell-based ELISA kit 1 hour after 5 Gy IR and the pSMC1data normalized with Glyceraldehyde-3-phosphate dehydrogenase (GAPDH).
SMC1 phosphorylation was significantly low in A-T`s PBMC (mean standard deviation [SD]: 0.075 0.034) in comparison to carriers (mean SD: 0.190 0.060) and healthy controls (mean SD: 0.312 .081), but unluckily could only discriminate A-T patients (Area Under the Curve -receiver operating characteristic [AUC-ROC]: 1.00, 1.00-1.00). This method in spite of rapidness and simplicity showed poor imprecision (22.49% coefficient of variation [CV] for intraday imprecision).
It seems pSMC1 assessment by in-cell ELISA can be used for detection of A-T patients, but it may not sensitive enough for identification of carriers. This ELISA test is very simple, rapid, and requires less than 2cc blood. Thus it may be proposed for the early differential diagnosis of A-T as an alternative method.

Myelodysplastic syndromes (MDSs) are a clonal bone marrow (BM) disease characterized by ineffective hematopoiesis, dysplastic maturation and progression to acute myeloid leukemia (AML). Methylation silencing of HRK has been found in several human malignancies. In this study, we explored the association of HRK methylation status with its expression, clinical parameters and MDS subtypes in MDS patients. To study the methylation status of HRK gene, we applied Methylation Sensitive-High Resolution Melting Curve Analysis (MS-HRM) in MDS patients, as well as healthy controls and EpiTect®PCR Control DNA. Real time RT-PCR was used for gene expression analysis. Methylation frequency in promoter region of HRK in patient samples was 20.37%. Methylation of HRK was significantly related to transcriptional downregulation (P=0.023). The difference in frequency of hypermethylated HRK gene was significant between good (10%) and poor (71.42%) cytogenetic risk groups (P= 0.001), advanced stage MDS patients (66.66%) in comparison with early stage MDS patients (2.56%) (P= 0.00), higher- risk MDS group (61.53%) and lower- risk MDS group (7.31%) (P= 0.00). HRK hypermethylation was associated with advanced- stage MDS and downregulation of HRK gene may play a role in the progression of MDS.

The secondary genetic changes other than the promyelocytic leukemia-retinoic acid receptor (PML-RARA) fusion gene may contribute to the acute promyelocytic leukemogenesis. Chromosomal alterations and mutation of FLT3 (FMS-like tyrosine kinase 3) tyrosine kinase receptor are the frequent genetic alterations in acute myeloid leukemia. However, the prognostic significance of FLT3 mutations in acute promyelocytic leukemia (APL) is not firmly established. In this study, the chromosomal abnormalities were analyzed by bone marrow cytogenetic in 45 APL patients and FLT3 internal tandem duplications (ITD) screening by fragment length analysis and FLT3 D835 mutation by melting curve analysis were screened in 23 APL samples. Cytogenetic study showed 14.3% trisomy 8 and 17.1% chromosomal abnormalities other than t(15;17). About 13% of the patients had FLT3 ITD, and 26% had D835 point mutation. FLT3 ITD mutation was associated with higher white blood cell count at presentation and poor prognosis. The PML-RARA translocation alone may not be sufficient to induce leukemia. Therefore, we assume that FLT3 mutations and the other genetic and chromosomal alterations may cooperate with PML-RARA in the development of APL disease.

The secondary genetic changes other than the PML-RARA fusion gene may contribute to the acute promyelocytic leukemogenesis. Chromosomal alterations and mutation of FLT3 tyrosine kinase receptor gene are the frequent genetic alterations in acute myeloid leukemia (AML). However, the prognostic significance of FLT3 mutations in acute promyelocytic leukemia (APL) is not firmly established.Methods and materials: Cytogenetic analysis of 45 APL patient's BM samples, FLT3 ITD screening by fragment length analysis and FLT3 D835 mutation by melting curve analysis in 23 APL samples was screened in this study. Cytogenetic study showed 14.3% trisomy 8 and 17.1% chromosomal abnormalities other than t(15;17). About13% of the patients had FLT3 internal tandem duplications (ITDs), and 26% had D835 point mutation. FLT3 ITD mutation was associated with higher white blood cell (WBC) count at presentation and poor prognosis. As the PML-RARA is not sufficient to develop APL, we assume FLT3 mutations and additional chromosomal alterations we found in this APL series may cooperate with PML-RARA in APL development.