مقالات رزومه دکتر سید حمیدالله غفاری

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.

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.

 Acute myeloid leukemia (AML) is described by the clonal expansion of myeloid blasts with abnormal differentiation. Considering the role of Toll-like receptors (TLRs) in inflammation induction and the effect of chronic inflammation on cancer development, investigating the state of TLRs’ expression in human malignancies has attracted scientists’ attention.

 In this study, 36 newly-diagnosed AML patients and 36 control samples were examined. The mRNA expression levels of TLR1/2/4/7/8 were measured in both groups using real-time PCR. The student’s t-test was utilized to compare gene expression levels between the two populations and the one-way ANOVA test was used to compare data among multiple subtypes.

All TLR gene expression levels were significantly up-regulated in patients compared to the control group (p<0.05). Positive correlations between different TLRs were observed as well. AML patients under the age of 55 showed significantly higher TLR1/2/4 expression in comparison with healthy individuals of the same age; a similar comparison in people above 55 also showed an elevated expression of TLR1/2/4/8. Male patients overexpressed almost all genes compared to healthy subjects; the levels of TLR1/2/4 were also higher in female patients. No difference was observed comparing blast percentages and FAB subtypes.

 By considering the results of this experiment, it seems that TLRs up-regulation in AML patients may contribute to the pathogenesis and development of the disease; however, more investigations are required to elucidate the exact roles of these receptors in AML. 

Minimal residual disease (MRD) is considered the greatest prognostic factor in acute lymphoblastic leukemia(ALL). MRD is a valuable tool for anticipating impending relapse and treatment response assessment. The objective ofthe present study was to investigate whether the detection of IgH gene rearrangement using polymerase chain reaction(PCR)-based GeneScan analysis could be a complementary method to monitor MRD along with the quantitative realtimePCR (qPCR). In this cross-sectional study, we valued the MRD levels, based on the GeneScanning analysis(GSA), and then compared the data with quantitative real-time polymerase chain reaction at different time points inperipheral blood (PB) samples of adult B-lineage ALL patients (n=35). The specific polymerase chain reaction (PCR)primers for IGH gene FR-1 and fluorescence-labeled J-primer were used and analyzed by capillary gel electrophoresison a sequencer. The results of this study were compared with the previously reported MRD results obtained by the IGHrearrangements allele-specific oligonucleotide (ASO) -qPCR methods. The total concordance rate was 86.7%, with a P<0.001. MRD results obtained by GSA and ASO-qPCR methodswere concordant in all diagnostic samples and samples on the 14th and 28th days of induction therapy. The results of these 2.5years’ follow-ups demonstrated a significant correlation between the two techniques (r=0.892, P<0.001). It seems that the PCR-based GeneScan analysis of IGH gene rearrangement detection may be a valuablemolecular technique to distinguish monoclonality from polyclonality. And also, it may be a precise tool to detect theresidual leukemic DNA in the PB follow-up samples of patients.

 The high mortality rate of Gastric Cancer (GC) is a consequence of delayed diagnosis. The early diagnosis of GC could increase the five-year survival rate among patients. We aimed to find a panel of microRNAs (miRNA) for the detection of GC in the early stages.

 In this case-control study, we selected consistently upregulated miRNAs from the results of 12 high-throughput miRNA profiling studies in GC. In the profiling phase, the differential expressions of 13 candidate miRNAs were analyzed by quantitative reverse-transcription PCR (qRT-PCR) in two pooled RNA samples prepared from the plasma of eight GC patients and eight matched controls. In the validation phase, significantly upregulated miRNAs from the profiling phase were further evaluated in the plasma samples of 97 patients with stage I-IV gastric adenocarcinoma and 100 healthy controls.

 In the profiling phase, six miRNAs (miR-18a, 21, 25, 92a, 125b and 221) were significantly upregulated in the GC patients compared to the controls (p<0.05). However, in the validation phase, only significant up-regulation of miR-18a, 21 and 125b was confirmed (p<0.05). A panel of miR-18a/21/125b was able to detect GC patients with stage I-IV from the controls (p<0.001; AUC=0.92, sensitivity=86%; specificity=85%). In addition, the panel could distinguish the early-stage GC (I+II) from the control group with an AUC of 0.83, a sensitivity of 83%, and a specificity of 75%.

 A panel of circulating miR18a/21/125b could be suggested as a potential biomarker for the early detection of GC.

Glioblastoma (GBM), the most aggressive and common form of glioma, accounts for over 13,000 death per year in the United States which indicates the importance of developing novel strategies for the treatment of this fatal malignancy. Although Arsenic trioxide (ATO) hinders the growth and survival of GBM cells, the requirement of concentrations higher than 4 μM for triggering apoptotic cell death has questioned its safety profile. Since the NF-κB signaling pathway plays a crucial role in tumorigenesis and chemo-resistance, targeting this oncogenic pathway may sensitize GBM cells to lower concentrations of ATO.

Anti-tumor effects of ATO as monotherapy and in combination with Bay 11-7082 were determined using MTT, crystal violet staining, Annexin V/PI staining and scratch assays. Quantitative reverse transcription-PCR (qRT-PCR) analysis was applied to elucidate the molecular mechanisms underlying the anti-tumor activity of this combination therapy.

Our results revealed that ATO and Bay 11-7082 synergistically inhibited the proliferation and survival of GBM cells. Also, it was revealed that NF-κB inhibition using Bay 11-7082 enhanced the inhibitory effects of ATO on migration of GBM cells via suppressing the expression of NF-κB target genes such as TWIST, MMP2, ICAM-1, and cathepsin B. Furthermore, combination treatment of GBM cells with ATO and Bay 11-7082 significantly induce apoptotic cell death coupled with downregulation of NF-κB anti-apoptotic target genes including Bcl-2 and IAP family members.

Altogether, these findings suggest that combination therapy with ATO and Bay 11-7082 may be a promising strategy for the treatment of GBM.

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.

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.

Daunorubicin (DNR) is capable of killing the human acute myeloid leukemia cells through apoptosis or necrosis with arresting cell cycle and various mechanisms. The response of AML cells to DNR associated with defect and or defiance in survival has been a consideration subject.
We have represented the transcription gene profile of some critical prosurvival proteins by qRT - PCR, including osteopontin (OPN), AKT1, mTOR, β - catenin, and NF - kB/RelB.
The U937 cells were treated with DNR with clinically achievable concentrations for MTT assay, annexin V (AV) /Propidium iodide (PI), and cell cycle analysis. QRT - PCR was performed, using primers of OPN, NF - kB/RelB, AKT1, mTOR, PTEN, and β - catenin.
The AV/PI assay displayed that DNR - induced death in cells was a dose - dependent and necrotic manner. Cell cycle distribution following treatment with DNR exhibited a relatively lower chromatin of S phase than untreated cells. OPN gene expression was significantly attenuated. NF - kB/RelB, mTOR, β - catenin, as well as PTENgenes showed unchanged or non - significant increase in expression. However, AKT1increased significantly.
U937 sensitivity to DNR could be due to the targeting of anti-apoptotic proteins in the transcriptional stages. The decrease in OPNlevels appears to play a significant role in the death of the observed by DNR.

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.

Colorectal cancer (CRC) is one of the most common cancers worldwide and considered to be one of the hassle in medical communities. CRC develops from precancerous polyps in the colon or rectum and is preventable and curable by an early diagnosis and with the removal of premalignant polyps. In recent years, scientists have looked for inexpensive and safe ways to detect CRC in its earliest stages. Strong evidence shows that screening for CRC is a crucial way to reduce the incidence and mortality of this devastating disease. The main purpose for screening is to detect cancer or pre-cancer signs in all asymptomatic patients. In this review, we holistically introduce major pathways involved in the initiation and progression of colorectal tumorgenesis, which mainly includes chromosome instability (CIN), microsatellite instability (MSI), the CpG island methylator phenotype (CIMP), and we then will discuss different screening tests and especially the latest non-invasive fecal screening test kits for the detection of CRC.

Interfering with cell proliferation and survival is a critical role for antineoplastic drugs leading to cell death through induction of apoptosis. Alternative treatments with herbal extracts offer insights into acute myeloid leukemia (AML) therapy. Parthenolide (PTL), an extract from feverfew, induces apoptosis in primary human leukemia stem cells (LSCs) and bulk leukemic cell populations. Osteopontin (OPN) preserves cell viability in response to anticancer agents and its receptors could be utilized for therapeutic targeting of cancer cells.
U937 cells were cultured in RPMI 1640 with concentrations of 2, 4, 6, 8, and 10 μM PTL for 20-24 hours for MTT assays. Apoptosis assays were performed with Annexin V-Alexa Fluor-488/PI as Annexin Vﳲ and Annexin Vﳲ to measure early and late apoptosis, respectively. Quantitative real-time PCR was used to measure OPN gene expression using the 2-∆∆Ct method. The PTL–treated cells were stained with FITC-CD38 antibody for flow cytometry analyses. Data were compared using one-way analysis of variance (ANOVA) by SPSS 19.
Parthenolide inhibited growth of U937 cells with IC25 and IC50 values of 4 and 5.8 µM, respectively. Death induction with PTL was apoptotic. Flow cytometry showed a significant decrease in the percentage of CD38 U937 cells in response to PTL. Osteopontin gene expression decreased in response to PTL.
PTL induced apoptosis and reduced OPN gene expression in U937 cells.

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.