Table of Contents

Original articles

diet, Physical activity, Marital Status and risk of Cancer: a Case Control Study of adults from riyadh, Saudi arabia .......................06Eyad Fawzi AlSaeed and Mutahir A. Tunio

Clinico-hematological Profile of 184 Patients with non-Hodgkin’s lymphoma: an experience from Southern Pakistan ...................11Sadia Sultan, Syed Mohammed Irfan, Anila Rashid, Saira Parveen and Neesha Nawaz

ambiguity of Whole Body PeT CT Scans in diagnosis of Co-existing Tuberculosis and Malignancy: is Histopathological Confirmation Mandatory? .........................................................................................................................................15Prekshi Chaudhary, Sweety Gupta, Nitin Leekha, Ravi S. Rajendra, Shiv S. Mishra, Vandana Arora, Sudarsan De and Sandeep Agarwal

epidemiology and Outcomes with Platinum-Based Chemotherapy in recurrent or Metastatic Carcinoma Cervix in a developing Country: experience from a Tertiary Oncology Centre in Southern india ......................................................................20K.C. Lakshmaiah, Aditi Harsh Thanky, D. Lokanatha, K. Govind Babu, Linu Jacob, Suresh Babu, A.H. Rudresha, K.N. Lokesh, L.K. Rajeev and Aparna Sridharmurthy

disclosure of adverse Cancer news: The Public’s Perspective in a Middle eastern Country .................................................................27Jamal Zekri, Mohamed E. El Sayed and Youssef Nauf

Second Primary Tumors associated with Breast Cancer: Kuwait Cancer Control Center experience ....................................................35Salah Fayaz, Gerges Attia Demian, Heba El-Sayed Eissa and Sadeq Abuzalouf

implications of Observer variation in Gleason Scoring of Prostate Cancer on Clinical Management: a Collaborative audit ...............41A. Harbias, E. Salmo and A. Crump

Squamous Cell Carcinoma of the Buccal Mucosa: a Single institute retrospective analysis of nodal involvement and Survival ...........................................................................................................................................................46Vivek Tiwari and Rakesh Mahawar

Clinical and Pathological Characteristics of Triple Positive Breast Cancer among iraqi Patients ..........................................................51Nada A.S. Alwan, Faisal H. Mualla, Munawar Al Naqash, Saad Kathum, Furat N. Tawfiq and Sana Nadhir

Pre-Treatment nutritional Status and radiotherapy Outcome in Patients with locally advanced Head and neck Cancers ...............61Amit Bahl, Arun Elangovan, Satinder Kaur, Roshan Verma, Arun Singh Oinam,Sushmita Ghoshal, Naresh K Panda

evaluation of BrCa1 large Genomic rearrangements in Group of egyptian female Breast Cancer Patients Using MlPa ...................................................................................................................................................................................64Ola M. Eid, Eman A. El Ghoroury, Maha M. Eid, Rana M. Mahrous, Mohamed I. Abdelhamid, Zahra I. Aboafya, Esmat A. Abdel Ghaffar and Amany H. Abdelrahman

Case reportsBrain Metastasis from Colorectal adenocarcinoma: a Case report ........................................................................................................70Jaroslav Nemec, Abdulsalam Alnajjar, Jasem Albarrak , Shaban A, Mariam Al Otaibi and Asit Mohanty

leiomyosarcoma of Penis: an aggressive and exceptionally rare entity ...............................................................................................73Vinita Trivedi, Muneer A, Rita Rani, Richa Chauhan, Usha Singh and Naveen Kuna

review articlesChemotherapy-induced febrile neutropenia in Solid Tumours ...............................................................................................................77Ayman Rasmy, Mohammed Al Mashiakhi and Amal Ameen

Conference Highlights/Scientific Contributions• NewsNotes ............................................................................................................................................................................................85

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

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

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64

Corresponding author: Ola M. Eid, PhD, Human Cytogenetics Department, Human Genetics and

Genome Research Division, National Research Centre, Bohouth Street, 12311 Dokki, Cairo, Egypt. Tel: + 20

100 177 5606; Email: olameid@hotmail.com

introduction Genomic rearrangements are mutational alterations

in the genome such as deletions, duplications, insertions, inversions or translocations (1). Genomic rearrangements represent polymorphisms which may be of neutral function, or may convey phenotypes through various mechanisms, for instance genes copy number alteration, genes disruption or fusion genes formation. The pathological situations occurred due to genomic rearrangements are known as genomic disorders (2).

Genomic rearrangements are gross DNA changes varying in size from thousands to millions of base pairs which may include groups of various genes. There is obvious difference between the large genomic rearrangements (LGRs) and the small scale gene mutations such as point mutations, indels. This difference concerns the size of the rearranged DNA, the mechanisms

abstract

introduction: Breast cancer is one of the most widespread cancers affecting women all over the world. In Egypt, it is considered to be the first cause of malignancies among female. BRCA1 Large Genomic Rearrangements (LGRs) have been reported in hereditary breast families and occurs in considerable proportion of cases in various populations.

Objective and methods: We investigated the incidence of BRCA1 LGRs in group of Egyptian females with breast cancer using Multiplex Ligation-dependent Probe Amplification (MLPA) assay.

results: Thirty six female breast cancer patients were included in this study. There were no BRCA1 LGRs

detected in the studied group of patients which does not coincide with other study that were done on a group of Egyptian female patients.

discussion and Conclusion: This variance may be due to the small number of the investigated patients in both studies, which is considered as a limitation. So, screening for LGRs of BRCA1 gene as well as other genes that may be involved in breast cancer such as BRCA2 and CHEK2 genes of a larger number of patients is recommended to get the actual prevalence of these gene in the Egyptian population to deliver a cost-effective primary approach for these patients.

Keywords: BRCA1, Large Genomic Rearrangements, Breast Cancer, Multiplex Ligation-dependent Probe Amplification (MLPA).

Original Article

evaluation of BrCa1 large Genomic rearrangements in Group of egyptian female Breast Cancer Patients

Using MlPaOla M. Eid1, Eman A. El Ghoroury2, Maha M. Eid1, Rana M. Mahrous1, Mohamed I. Abdelhamid3,

Zahra I. Aboafya2, Esmat A. Abdel Ghaffar2, Amany H. Abdelrahman2

1Human Cytogenetics Department, National Research Centre, Egypt 2Clinical Pathology Department, National Research Centre, Egypt

3General Surgery Department, Zagzeg University, Egypt

by which the rearrangements are formed as well as the mechanisms of the conveying of phenotypes. Point mutations generally mirror faults of DNA replication and/or repair, while the LGRs are commonly caused by different mechanisms induced by the genomic structural features (1). LGRs can be recurrent with a common size and constant breakpoints. They also can be non-recurrent with various sizes and distinct breakpoints for each situation. The non-recurrent rearrangements have a common genomic region-of-overlap, the smallest region of overlap (SRO), which embraces the locus associated with the conveyed genomic disorder. Non-allelic homologous recombination

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(NAHR), non-homologous end-joining (NHEJ) and the Fork Stalling and Template Switching (FoSTeS) models are the suggested mechanisms for genomic rearrangements. When these mechanisms take place in germ cells, the rearrangements cause genomic disorders. But when it occur in somatic cells, the genomic rearrangements lead to disorders such as cancer. Non-allelic homologous recombination (NAHR) causes most of the recurrent rearrangements that have a common size, display clustering of breakpoints, and recur in many individuals. NAHR is commonly mediated by low-copy repeats (LCRs) with recombination hotspots, gene conversion and apparent minimal efficient processing segments (2).

Breast cancer is one of the most widespread cancers affecting women all over the world (3). In Egypt, it is considered to be the first cause of malignancies among female, occurring in 18.9% of total cancer cases. It is represents 15% of cancer deaths in Egyptian females (4,

5). Positive family history and genetic factor are main risk factors for developing breast cancer (6).

Inherited breast cancer is a highly heterogeneous genetic disease. LGRs have been reported in hereditary breast families and occurs in considerable proportion of cases in various populations (7). Deletions or insertions of large genomic sequences within a coding region lead to out-of-frame translation consequently formation of a mutant peptide of abnormal structure and/or function (8). LGRs of BRCA1 reported in up to 33% of all pathogenic mutations in various populations, while LGRs in BRCA2 are less frequently described (7, 9).

There are numerous BRCA1 germline rearrangements with well characterized breakpoints (10). They are dispersed all over the gene in the form of deletions, duplications, triplications or combined deletion/insertion. The BRCA1 gene contains high density of intronic Alu repeats. As well, it has a duplicated promoter region containing a BRCA1 pseudogene which favor the occurrence of “hot spots” which promote unequal homologous recombination events (11, 12). Globally, there are 45 different BRCA1 LGRs that have been described, including deletions and duplications of one or more exons (7). Alu elements ranging from 0.5 kb to 23.8 kb in size represent about 41.5% of the intronic sequences of BRCA1 and are found throughout the whole gene (13). Alu sequences are considered a genomic instability factors as they are accountable for recombinational “hot spots” in certain genes. Moreover in most of the well characterized rearrangements described, Alu repeat elements are implicated in the recombination event. Therefore, LGRs are less frequent in the BRCA2 gene, possibly because of the less incidence of Alu sequences (17%). (7)

Pseudogenes are counted as non-functional “relatives” of known genes. In another words, they are genes that either have lost their protein-coding ability or are no longer expressed in the cell. They are deemed as important cause of unequal recombination within the coding region of particular genes (7). It was reported that the first exons of the BRCA1 gene are substituted by those of the BRCA1 pseudogene, ψBRCA1. Furthermore, the existence of a duplication involving most of BRCA1 exons 1 and 2 and the detection of two different recombination events involving homologous regions situated in the BRCA1 gene and ψBRCA1, respectively, had led to assume that these regions are potent “hot spots” for recombination (12).

Moreover, tandemly arranged short sequence repeats may facilitate gross chromosomal deletions and/or insertions. It was reported that recombination involving tandemly arranged short sequence repeats causes the BRCA1 exon 5 deletion in hereditary breast cancer of German families (8).

LGRs mostly missed by the diagnostic PCR-based methods which are qualitative rather than quantitative methods as well as they are unable to identify partial or complete exon losses or gains (7, 14). Multiplex Ligation-dependent Probe Amplification (MLPA) was initially designated in 2002 by Shouten et al. (15). It relies on sequence-specific probe hybridization to genomic DNA, amplification of the hybridized probe and semi-quantitative analysis of the resultant PCR products (16). It is a multiplex PCR technique that identify abnormal copy numbers of up to 50 different genomic DNA or RNA sequences distinguishing sequences differ in only one nucleotide (17). In MLPA, the MLPA probes that hybridize to the target sequence are amplified, not the target sequences itself. A single pair PCR primers is employed in MLPA amplification. The amplified products are then analyzed by capillary electrophoresis. Aberrant copy numbers are detected by comparing the achieved peak pattern of the sample to that of reference samples (18). MLPA is mainly used as a screening method to detect rearrangements. It is a reasonably inexpensive, sensitive, easy and high-throughput method. However, MLPA has few drawbacks, such as false-negative results when probes are designed outside the region implicated in the rearrangement. Moreover occasionally, MLPA may produce a false-positive deletion due to occurrence of a point mutation within the sequence of MLPA probe (19). The implementation of MLPA has facilitated the screening of genomic rearrangements in BRCA1 (7, 20).

The aim of this study is to investigate the incidence of BRCA1 large genomic rearrangements in group of Egyptian females with breast cancer.

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BRCA1 LGRs in Egyptian Female Breast Cancer Patients Using MLPA, Ola M. Eid, et. al.

Patients and Methods This study was conducted at the National Research

Centre, Egypt and was approved by its Medical Ethical Committee. Thirty-six female breast cancer patients were included in this study. Informed consents were obtained from the patients. Thirty-two patients were infiltrating duct carcinoma and only four were invasive lobular carcinoma. All the infiltrating duct carcinoma were of grade II except two patients were of grade III. Only 8 out of the 30 patients of grade II infiltrating duct carcinoma have auxiliary lymph node metastasis.

Peripheral blood sample (5ml) from the patients and the reference samples (one reference for 7 patients sample with minimal 3 references per test) was collected on K2-EDTA in vacutainer tube for DNA. DNA extraction from 0.2 ml of the peripheral blood lymphocytes was carried out using QIAamp DNA Mini Kit (Qiagen, Germany) according to the manufacturer’s instruction. The quality and quantity of the DNA samples were evaluated using the NanoDrop spectrophotometer.

LGRs evaluation were carried out using SALSA MLPA P239 BRCA1 region probemix, according to the manufacturer’s instruction (MRC-Holland, Amsterdam, Netherlands). This probemix characterizes BRCA1

deletions/duplications. DNA denaturation and overnight hybridization of the MLPA probemix were done, followed by probe ligation and amplification in the next day. Separation of amplified products were done using Genetic Analyzer ABI 3500 (USA). Interpretation of the results was done utilizing Coffalyser. Net software (MRC-Holland). The generated data were (Intra and inter-sample) normalized. Ratios less than 0.75 were considered as deletion, between 0.75 and 1.30 as normal and more than 1.30 as duplication.

results MLPA assay did not detect any LGRs in BRCA1 gene in

the studied group of patients. Figure 1 shows a ratio chart of MLPA result for the BRCA1 gene of one of the breast cancer patients. The average ratios were within the 0.75-1.3 range demonstrating absence of exon deletions or duplications of the BRCA1 gene in all samples analyzed.

discussion and ConclusionThe prevalence of any mutation in a certain population

should be known before the final recommendations are reached concerning the routine workout for this rearrangements. Numerous reports of various populations have proven the worth of screening for BRCA1

figure 1. a ratio chart of MlPa result of one of the breast cancer patients using SalSa MlPa P239 BrCa1 region probe mix. The black spots represent MlPa probes, the upper blue line indicates a peak ratio of 1.3 and any probes above this line represent a duplication, the lower red line indicates a peak ratio of 0.75 and any probes below this line represents a deletion, and the probes between the two lines are considered as normal two copies. The chart is showing no deletion or duplication as all the spots are between the both cut-off lines in the ratio chart.

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rearrangements. In populations where there are highly prevalence of BRCA gene mutations, initial screening for them may be a cost-effective primary strategy (7).

The prevalence of BRCA1/BRCA2 mutations in patients with breast cancer is widely varied depending on the age and the gender of the patient, the tumor histology and the family history. LGRs account for 12-18% of all BRCA1/2 mutations (21, 22). BRCA mutation carriers possess a great risk of breast cancer by age 70 ranging of 47-66%. Moreover, females with BRCA mutation-associated breast cancer also have high risk of other or secondary malignancies, such as pancreas cancer, gastrointestinal cancers and melanoma. BRCA1-associated breast cancers are mostly of high grade (22).

There is a diversity in the incidence of BRCA1/BRCA2 rearrangements present in various ethnic groups and populations. So, identification of BRCA rearrangements in a population is of high importance as in some populations the incidence of large genomic rearrangement in form of deletions or duplications in either BRCA1 or BRCA2 is significant (23). A prevalence of 2.1% for BRCA1 LGRs has been noticed in Spanish hereditary breast cancer families testing negative for point mutations and small insertions/deletions in BRCA1 and BRCA2 (24). Where in Northern Finland, a large deletion of exons 1A-13 in BRCA1 gene was detected, representing 14.3% of the Finnish population (25). In certain European and American populations, BRCA1/2 LGRs have shown obvious founder effect. BRCA1 LGRs, exon 9–12 deletion, was counted as a mutation in Mexican population (26). However, the incidence of BRCA1/BRCA2 LGRs in Asians is assumed to be low (23). Nevertheless, studies carried out in these populations are limited. Several deletions and duplications have been described in Singaporean (27), Korean (28), Malaysian (29) and Chinese (30) populations. Moreover, BRCA1 and BRCA2 LGRs are doubtfully contribute to the etiology of breast cancer in Sri-Lanka (23). Nevertheless, MLPA was suggested as an initial screening method for mutations in the BRCA1 gene (31).

BRCA mutations in Arab countries were scarcely investigated. A study of 35 Sudanese patients suggested a significant role of BRCA1 and BRCA2 genetic mutations in breast cancer of males and young women (32). A Tunisian study done on 36 selected patients, found 4 BRCA1 and 2 BRCA2 frameshift mutations (33). A high incidence of BRCA1 mutations were observed in familial Algerian female patients as well as in Moroccan female patients (34, 35). Deleterious BRCA1 and BRCA2 mutations were found in 12.5% of Lebanese patients in a group of 72 females with early onset breast cancer and/or positive family history (36). However, in another study, the incidence of deleterious BRCA mutations was lesser

than anticipated and does not support the assumption that BRCA mutations are the only cause of the high incidence of breast cancer in young females of Lebanese and Arab descent (37). Four BRCA1 exons and one BRCA2 exon were assessed in 60 selected and referred Egyptian females and showed a mutation prevalence rate of 25% (5). Moreover, LGRs were observed in 4 out of 22 high-risk Egyptian female breast cancer patients (18.2%) providing the first evidence that LGRs of BRCA1 exist in the Egyptian population (6). However, there was lack of association between BRCA1 and BRCA2 variants in 100 breast cancer Saudi Arabian patients (38). The variability of outcomes of these studies depends on the selection criteria of the patients and referral patterns.

LGRs of BRCA1 gene in Egyptian population have not been extensively studied. Our results coincides with the Sri-Lanka study of LGRs of BRCA1 gene in breast cancer patients (23). However, there were evidence of the presence of BRCA1 LGRs in the Egyptian population (6). This variance may be due to the small number of the investigated patients in both studies, which is considered as a limitation. So, screening for LGRs of BRCA1 gene as well as other genes that may be involved in breast cancer such as BRCA2 and CHEK2 genes of a larger number of patients is recommended to get the actual prevalence of these gene in the Egyptian population to deliver an economical primary approach for these patients.

funding

This study was funded by the National Research Centre, Egypt.

Conflict of interest

The authors declare that they have no conflict of interest.

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