Table of Contents

Original articles

Pattern of Karyotypic aberrations in Pakistani Patients with de novo acute Myeloid leukemia ..........................................................06Syeda Alia Abbas, Sadia Sultan, Sana Ashar, Syed Muhammad Irfan

Tinospora Cordifolia induces Cell Cycle arrest in Human Oral Squamous Cell Carcinoma Cells ...........................................................10Parveen Bansal, Manzoor Ahmad Malik, Satya N Das, Jasbir Kaur

detection Mutations of JaK2 exon 12 in Patients with JaK2 (v617f)-negative Myeloproliferative disorders .....................................15S. Z. Makani, N. Parsamanesh, S. Mirzaahmadi, M. Hashemi, F. Shaveisi-Zadeh, N. Mansouri, M. Ghazi, A. Movafagh

Hepatocellular Carcinoma Peritoneal Metastasis: role of Cytoreductive Surgery and Hyperthermic intraperitoneal Chemotherapy (HiPeC) ......................................................................................................................................................20John Spiliotis, Georgios Nikolaou, Nikolaos Kopanakis, Dimitra Vassiliadou, Alexios Terra, Elias Efstathiou

The effect of dose-volume Parameters on Central nervous System relapse in Pediatric Patients with acute leukemia receiving Prophylactic Cranial irradiation ............................................................................................................24Zeliha Guzeloz, Ayse Nur Demiral, Fatma Eren, Mehmet Adigul, Ahmet Ergin Capar, Handan Cakmakcı, Sebnem Yilmaz, Ozlem Tufekci, Hale Oren, Riza Çetingoz

long-term results of Post-operative Pelvic image guided intensity Modulated radiotherapy in Gynecological Malignancies ........30Rashi Agrawal, Sowmiya Prithiviraj, Dinesh Singh, Vaishali Zamre, Sandeep Agrawal, Arun Kumar Goel, Kanika Gupta, Bala Subramanian

epidemiology and Survival analysis of Gastrointestinal Stromal Tumors in lebanon: real-life Study from a hospital tumor registry, 2000-2015 ....................................................................................................................38Elie El Rassy, Fadi Nasr, Tarek Assi, Toni Ibrahim, Nathalie Rassy, Joseph Bou Jaoude, Marcel Massoud, Georges Chahine

Case reports

Giant Primary Sinonasal Mucosal Melanoma: a rare Malignancy ..........................................................................................................43Mehtab Alam, Mohd Aslam, Piyush Kant Singh, Shahab Farkhund Hashmi, Syed Abrar Hasan

Sarcomatoid Carcinoma of the Maxilla: a Case report with literature review ......................................................................................48Lahcen Khalfi, Yassine Ziani, Mouna Kairouani, Odry Agbessi, Mohammed Kamal Fiqhi, Alae Guerrouani, Karim El Khatib

neuroendocrine Carcinoma of Gall Bladder: a rare Presentation with review of literature .................................................................51Amit Gupta, Parvez Ahmed, Prashant Durgapal, Pooja Kala, Shalinee Rao, Rajesh Pasricha, Sanjeev Misra

Clear Cell variant of Calcifying epithelial Odontogenic Tumor: a rare Clinical entity ............................................................................55Husain Sabir, Subhash Kumbhare, Saurabh Redij, Namrata Gajbhiye

Hodgkin’s lymphoma as a Multiloculated lung Cavity lesion ................................................................................................................61Aisha M. Al-Osail, Hind S. Al-Saif, Mashail M. Al-Hajri, Hajer M. Al-Zuhair, Deemah A. Al-Abdulhadi, Emad M. Al-Osail, Sarah M. Al-Hajri

Breast adenoid Cystic Carcinoma: a rare Case .......................................................................................................................................66Lamiae El Amarti

review articles

industrial Pollutants and nasopharyngeal Cancer: an Open Question ....................................................................................................70Roberto Menicagli, Gianni Bolla, Laura Menicagli, Anastassia Esseiridou

Conference Highlights/Scientific Contributions

• Highlightsofthe1stCombinedGulfCancerConference,CancerAwareness:RealityandAmbition,2-3April2017,Kuwait ........75

• NewsNotes ............................................................................................................................................................................................84

• Advertisements .....................................................................................................................................................................................88

• ScientificeventsintheGCCandtheArabWorldfor2017 ..................................................................................................................89

1515

Original Article

detection Mutations of JaK2 exon 12 in Patients with JaK2 (v617f)-negative Myeloproliferative disordersS. Z. Makani1, N. Parsamanesh2, S. Mirzaahmadi1, M. Hashemi3, F. Shaveisi-Zadeh4, N. Mansouri4,

M. Ghazi5, *A. Movafagh4

1Department of Biology, Zanjan Branch, Islamic Azad University, Zanjan, Iran 2Department of Molecular Medicine, faculty of Medicine, Birjand Medical University, Birjand, Iran. 3Department of Molecular Genetics, Tehran Medical Branch, Islamic Azad University, Tehran, Iran.

4Department of Medical Genetics, School of Medicine, Cancer Research Center, Shohada Referral Hospital, Shahid Beheshti University of Medical Sciences, Tehran- Iran. 5Department of Microbiology,

School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran.

abstract:

Background: Mutations in exon 12 of JAK2 gene are detected as clonal markers in hematopoietic lineages in myeloproliferative disorders (MPNs). Our aim was, to study the relation between N542-E543del mutation of JAK2 gene and myeloproliferative neoplasms in V617F-negative patients.

Patients and Methods: DNA specimen from 34 patients and 44 healthy controls were genotyped using ARMs-PCR method. We analyzed exon 12 JAK2 aberration in 34 myeloproliferative cases to be readily detected by both ARMS-PCR and DNA analysis regardless of whether peripheral blood or bone marrow cells was manipulated as the origin of RNA.

results: In this case-control study, there was no significant difference in Pearson chi square analysis

between the patients and control groups in genotype distribution of the frequency of single nucleotide polymorphism rs7869668 of JAK2 exon 12 (P > 0.05). Also, gene detection finding showed that the patients were negative of JAK2-V617F mutation.

discussion and Conclusion: Present finding on a small number of patients diagnosed of various categories of MPDs revealed and needs more investigation and data for the prevalence and the incidence of the JAK2–V617F mutation. However, the clinical and genotyping of finding a disorder and non-significant correlation between patients and control group in this study in such a small fraction of the patients is unknown.

Keywords: Genotype; JAK2 V617F; Exon 12; Myeloproliferative Disorders

Corresponding author: Abolfazl Movafagh, PhD, Department of Medical Genetics, School of

Medicine, Cancer Research Center, Shohada Referral Hospital, Shahid Beheshti University of Medical Sciences, Tehran- Iran, Fax: +98 (21) 2240067-

982123872572, 09121307881; Tel: +98-381-3361001.

Email: movafagh.a@sbmu.ac.ir

introductionThe history of Myeloproliferative neoplasms (MPNs)

started in 1951, where William Dameshek called the terminology myeloproliferative disorders (MPD) as a clinic and pathologic classification were established as (Di Guglielmo syndrome), polycythemia vera (PV), chronic myelogenous leukemia (CML), essential thrombocythemia (ET), primary myelofibrosis (PMF), and erythroleukemia (1).

The World Health Organization (WHO) 2008 category System for Myeloproliferative Neoplasms, accepted and approved mutation of JAK2 in myeloproliferative disorders. This acquired mutation is characterized by a nucleotide 1849 in exon 12 at G to T transversion mutation for the JAK2 gene, addressing to valine substitution to amino

acid phenylalanine at site 617 (V617F) of the JAK2 gene expression (2).

Many genetic analyses exhibited that a patient of myeloproliferative mutation with a normal blood cell complete count result showed for the JAK2-V617F and

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The JAK2 (V617F)-negative gene in Myeloproliferative Disorders, S. Z. Makani, et. al.

not available for the JAK2 exon 12 and 13 mutation and the BCR-ABL (p210) gene translocation (2).

Ikeda K, in 2016 reported that Myeloproliferative neoplasms (MPNs) are activated by a characteristic of the JAK-STAT pathway due to driver mutations including JAK2 V617F (3). The morphology data of JAK2 disorders in blood sample of polycythemia vera (PV) patients with exon 12 mutation and PV patients with JAK2-V617F also was reported (4).

The site of gene JAK2 V617F are located on 9p24 chromosome in exon 12 and occurring in most of the MPN. The downstream region of JAK2, evaluation of phosphorylation of STAT1 and STAT5 can increase megakaryopoiesis or erythropoiesis in the gene expression profile (5).

The discovery of the JAK2 V617F mutation and recent progress in the understanding of the genetic basis of the essential thrombocythemia (ET), Philadelphia (Ph.) chromosome-negative, myeloproliferative neoplasms (MPNs), myelofibrosis (MF), and polycythemia Vera (PV) (6) has made a significant contribution to understanding the pathogenesis of myeloproliferative neoplasms (7). The JAK2 (V617F) mutation is identified in nearly 95% of patients with polycythemia Vera (PV), with over than 50% of those having primary myelofibrosis (PMF) or essential thrombocythemia (ET) (8).

Among the patients of PV not diagnosed for the V617F mutation, several mutations in exon 12 is found in a significant number of cases. More than 10 different sequence variations have been found in exon 12 of the JAK2 location, most of those present in the region among codons 536 and 544(9-13) like N542-E543 del.

JH2 auto-inhibitory domain in exon 14 of JAK 2 consist of V617 mutation. Recent study showed that JAK2 mutation in exon 12 (V617 mutation) negative patient PV. The novel mutation reported such as N542-E543del and E543-D544del JAK2 617 mutation in exon 12 have been exhibited in the most of the patient of PV and increase the platelet and white blood cell. But some patient with myeloproliferative disorder haven’t mutation JAK2 in exon 12 (8).

In this research work, we investigate N542-E543 del mutation in patients with JAK2 (V617F)-negative myeloproliferative disorders.

Methods

Study design and Sampling

Subjects of the present study were 34 fresh and diagnosed patients of MPDs (female/male: 14/20, age

mean: 60.1 years, range: 25–88 years). The patients were chosen from admission clinic at Babol, Mazandaran, Iran from March 2014 and June 2015. We studied 34 patients with JAK2 (V617F)-negative myeloproliferative disorders as well as 44 normal control, were consistent with Hardy Weinberg equilibrium. This information was supported by the establish ethics members University of Zanjan. The procedures and samples were collected from patients after informed consent was taken in agreement with the Declaration of Helsinki 1964, as re-reported in 2013 http://www.wma.net/en/30publications/10policies/b3/) and the World Health Organization (WHO) criteria 2008 (1).

extraction of dna electrophoresis on agarose gel

Patients’ specimen DNA were taken from BM aspirates or PB using the QIAamp DNA Blood Mini Kit (Qiagen). The JAK2–V617F mutation negative were extracted based in accordance with Baxter et al. (14).

JaK2v617f Mutation analysis

analysis of PCr procedure

The PCR-product output was extracted in 1.6% ethidium bromide (EtBr)-stained agarose gel electrophoresis. Little amount of 16 µL of PCR product was used to the gel. Voltage of 120 V for 2 minutes and then 85 V for 26 minutes were applied for output differences. This genomic marker molecule sample (100 bp, Fermentas, Germany) were elaborated as a marker size. By electrophoresis, photograph was selected of UVI doc gel electrophoresis protocol (England). Particularly, the primers used for Fig 1.

To amplify exon 12, using the software Gene Runner, a forward and reverse primer pairs were designed. Sequences amplified during the PCR, the exact sequence of exon 12 and the primer binding sites exhibited in Fig 1.

Statistical analysis

After Analysis JAK2V617F Mutation, the results employed with t-test for standard variation and the chi-square test for classification variables as established. These association of the results were also tested using

figure 1. Sequence of exon 12 and the primer binding sites.

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G. J. O. Issue 24, 2017

a multivariate logistic regression that selected overall important variation. An association was expressed as odds ratio (OR) with the corresponding 95% confidence interval (CI). A variable was considered significant when p < 0.05. The analysis was performed using SPSS Version 13.

results The gene JAK2 in exon 12 (128 bp) and part of intron

12 and 13, was amplified by PCR machine. This sequence was amplified on agarose gel electrophoresis 2% as shown in Fig 2, 420-bp fragments by gel imaging devices.

direct sequencing

Nucleotide sequencing is a good way to accomplish this considering the wide variety of mutations that have been discovered in JAK2 exon 12 (15).

For the present study of patient’s specimen, allele-specific polymerase chain reaction (PCR) for the detection of JAK2-V617F were elaborated. Polymerase Chain Reaction product of diagnosed were extracted by Concert Rapid PCR Purification device (BRL Gibco, England and Lark Technologies, Inc). service (http://www.cogenics.com) were elaborated for sequencing detection of the pure Polymerase Chain Reaction products. For this purpose,

PCR products were sequenced and sequences were analyzed by Rapid PCR Purification System. Prevalence of JAK2–V617F mutation in patients with MPDs exhibit the figure 3 below position 542 and glutamic acid position 543 encoding the asparagine position JAK2 protein shows that the removal of these two sites, N542-E543del.

The frequency of single nucleotide polymorphism rs7869668 of JAK2 exon 12 in patients and controls are as allele A 48%, G 52% and corresponding to controls allele A 49%, G 51%. The result of statistical analysis exhibit P > 0.05 which shows non-significant. The data of this analysis summaries in Fig 4.

discussion and Conclusion

Current research in the information of the gene sequences of the essential thrombocythemia (ET), Philadelphia (Ph) chromosome-negative myeloproliferative neoplasms (MPNs), myelofibrosis (MF) was led to the identification of Janus kinase 2 (JAK2) and polycythemia vera (PV), as a possible therapeutic specimen for these disease.

The role of JAK/STAT signaling in normal hematopoiesis as well as genetic abnormalities associated with myeloproliferative and myelodysplastic syndromes previously reported (3,16).

The study of JAK2 suggests that mutation of V617F do not act as important function in the increase rate of disease for multiple myeloma (MM) and that another not understood system may support constitutive JAK2 activation, especially in IL-6-independent MM cellular mechanism (17). It is corresponding to our result that mutation of substitution of valine to phenylalanine at 617 amino acid position did not diagnosed. Including the JAK2V617F-negative, a number of fourteen genes

figure 2. PCr product on agarose gel at a concentration of 2%, wells containing M markers 50 bp PCr product of 420 bp in length.

figure 4. Genotype frequency of rs7869668 between patients and normal controls

figure 3. PCr product sequences in the coding region of n542-e543del; is marked with yellow box.

18

The JAK2 (V617F)-negative gene in Myeloproliferative Disorders, S. Z. Makani, et. al.

GADD45B, (TRIB1, C13orf18, CISH, MAFF, SOCS3, ID2, TNF, KLF5, TAGAP, HRH4, LAMB3, PIM1, and CCL3) exhibited a mutation evaluation analysis. Of these set of sample, SOCS3, SOCS2, CISH, and genes of PIM1, all involved in JAK-STAT signaling pathway, presented a lower gene expression (9,18).

A number of acquired mutations targeting JAK2 exon 12 is available in these patients including the polycythemia vera, neoplasm myeloproliferative, which decrease the much number of JAK2V617F mutations (19). This malformation in the JAK2 expression binding of the SH2 domain and JH2 occur among domain psudokinase although not real, but can the same mutation V617F, changing causes in the shape of JH2 domain (16). All together the mutations in exon 12 mutations N542-E543del the more common mutations in the gene expression (19). Overall many studies round the world with the relationship between mutations in exons 12 JAK2 and myeloproliferative neoplasms has been reported (20).

In the year 2011 Scott and his colleague were investigated cases of exon of 12 JAK2 mutation shows with an oncological mutation including for the development of polycythemia vera; these are recognized by erythrocytosis, with an increased hemoglobin and hematocrit parameter (20). In contrast with the study of Scott and colleagues (20), mutation in exons 12 including N542-E543 del mutation was not observed in any patient of our investigation.

In the year 2007 Williams et al reported that the pathophysiology appearance for exon 12 of JAK2 gene in the benign myeloproliferative disorder were increasing red cells, these are developing pathogen diagnose covering V617F JAK2 and JAK2 between exon twelve mutations. In opposite, myeloproliferative genetic aberrations were not incorporated with erythrocytosis, but separation originally with the appearance of myelofibrosis, osteosclerosis disease, extramedullary, and thrombocytosis (21).

In 2007 Percy et al, reported the exon 12 of JAK2 abnormality was found in 26% of cases in less plasma erythropoietin (Epo), most of cases have Epo-self erythroid progeny. Furthermore, IE patients showing for both of laboratory finding with the available of a JAK2 malformation (22).

Mutations in sequence gene twelve were found in different parts of JAK2V617F-negative IE or polycythemia vera cases with almost targeted the non-lymphoid lineages. However, the earlier oncologic information was different from V617F-positive cases, myelofibrotic transformation and thrombotic development during diagnosis are prone to classical JAK2 V617F-positive PV cases (23).

In 2007 Pitra and colleagues reported, different somatic mutations of exon 12 JAK2 could be diagnosed with a MPDs malignancy classified as erythrocytosis. Furthermore, a genetical pre disposition with acquired JAK2 mutation are familiar of classification of MPDs malignancy (24-27).

In summary, our data on a small number of patients diagnosed from different categories of MPDs confirmed and needs more investigation and more data of the prevalence and the incidence of the JAK2–V617F mutation.

The results of present investigation with JAK2-V617F mutation could be helpful for healthcare gaudiness for approaching patients with myeloproliferative disorders. However, the clinical and genotyping of detecting mutations with non-significant correlation results in this study in such a small fraction of population is unknown. Future research will be essential with the same project with higher number of patients or about the possible relation between the hematological malignancy prognosis and the MMPs SNPs would be a contribution to the literature.

acknowledgementThis research project is part of thesis results of Zahra

Makani MSc scholar. The presenter search work was conducted with funding from the office for Research at the Zanjan Branch, Islamic Azad University, Zanjan, Iran. All authors declare that there is no conflict of interests.

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