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دراسة جزيئية ومناعية كيميائية لانسجة عنق الرحم المصابه بفايروس الورم الحليمي البشري == Molecular and Immunohistochemical Study of Uterine Cervical Tissues Infected with Human Papilloma Virus
Author name:
الهام جواد كاظم بلال
Supervisor name:
ازهار عمران لطيف الذهب
General topic:
Biology
Specific topic:
Microbiology - Viruses
Degree:
Doctorate
University:
University of Babylon - College Of Science - Department Of Biology
Language:
English
University location:
Babylon
First pages:
24T3054 - p.pdf
Abstract:
Human papillomavirus (HPV) is a DNA virus. It is the main etiological factor for progress of cervical precancerous and cancerous lesions. In order to develop a cancer from dysplasia to invasive carcinoma, a series of cellular changes should occur. As genital HPV carries oncogenes involving in these critical changes, currently HPV has been considered the most important risk factor of cervical cancer.This study was designed as case control study. In the retrospective part of these study a total number of (56) formalin - fixed, paraffin embedded cervical tissues was involved were submitted to ISH and IHC technique 27 with Cervical Carcinoma (CC) and 16 with benign cervical tumors. Thirteen (13) apparently healthy cervical tissues were used as a control group .The age of this individuals (patients and control groups) was ranged between 23 and 73 years. Samples collected from the pathological archives of different general hospital as well as many private laboratories. Specimens belong to the period from January 2009 to September 2015. After sectioning of these cervical biopsies and staining by hematoxyline and eosin, a final definitive histopathological diagnosis was done by a consultant histopathologist.In the cross - section part of this study seventy two female patients taken from different general hospitals. Cytological examinations were performed for their Pap - smeared and a final definitive cytopathological diagnosis was done by a consultant histopathologist. According to their main cytological finding cervical cellular scrapes were divided into three cytological groups : cervicitis with squamous intraepithelial lesions, cervicitis without squamous intraepithelial lesions and healthy group. The DNA isolated from a representative cytological specimen was investigated by real - tim PCR analysis for the detection and genotyping of high/risk HPV DNA.The obtained results are summarized as follows : - The age range distribution mostly in age group (23 - 73 years), the most affected age group in malignant and benign cervical tumor was (52 - 67) , (36 - 51) years which constituted 55.6%(15 out of 27 cases), 50% (8 out of 16 cases) with mean age (54 ± 11.22), (38.8 ± 9.85) respectively, while (20 - 35 years) is the most affected age group in control cases were constituting 53.8% (7 out of 13 cases) with mean age (35.5 ± 8.64 ) . - The most common histopathological type among all studied archived cervical tissues was squamous cell carcinoma (66.6%), followed by the adenocarcinoma (33.3%). - The well differentiated grade of CC constituted 52%, whereas those with moderately and poorly differentiated grades CC constituted 37%and 11% respectively. - The percentage of HrHPV31/33 DNA was detected as follows : in 55.6 % of tissues from cervical cancers, 43.7% in the tissues from benign cervical tumors, while, no HrHPV31/33 positive - ISH reaction was detected in healthy cervical tissues of the control group. - The percentage of LrHPV6/11 DNA was detected as follows : in 33.3 % of tissues from cervical cancers, 31.3% in the tissues from benign cervical tumors, while, the percentage of LrHPV6/11 show negative - ISH reaction in healthy control group of cervical tissues. - The highest positive result for ISH of HPV31/33 found in squamous cell carcinoma 72.2% and in adenocarcinoma was 22.2% while the percentage of HPV6/11detection according to cancer type was 50% found in squamous cell carcinoma while 0.0% in the type of adinocarcinoma. - The highest percentage of HPV31/33 detected in relation with tumor grade depending on the differentiated of cells was 70% (7 out of 10 cases) show in moderately differentiated, followed by 57.1% (8 out of 14 cases ) in well differentiated , and none of poorly differentiated showpositive signal . - The highest percentage of HPV6/11 detected in relation with tumor grade depending on the differentiated of cells was 42,9 % (6 out of 14 cases) show in well differentiated, followed by 30% (3 out of 10 cases ) in moderately differentiated , and none of poorly differentiated show positive signal . - The highest percentage of co - infection with High risk HPV31/33 and Low risk of HPV6/11 types was (22.2%) in malignant cervical tumor group. While in benign cervical tumor and healthy control was 18.8% and 0.0% respectively. - The highest percentage of HPV DNA detection by Rt - PCR was 80% (16 out of 20 cases) within group of cervicitis with SIL, 16.7% (5 out of 30 cases) in group of cervicitis without SIL, while none of control group shoes positive result. - Phylogenetic tree analysis image, results that local HPV amplicon(S1, 3, 5, 7, 9, 12, 14, 15, 16, 17, 18, 20) show closed related to NCBI - Blast HPV type 16 (KMO58666.1), the local HPV isolates (S2, 4, 6, 11 and 19) show closed related to NCBI - Blast HPV type 18 (KC470225.1), whereas the local HPV isolates (S10 and 13) show closed related to NCBI - blast HPV type 33 (EU918766.1), the local HPV isolates (S8) show closed related to NCBI - Blast HPV type 45 (KC470260.1). - Retinoblastoma protein overexpression was detected by IHC test in 59.3% of malignant cervical tumors, 43.8% of benign cervical tumors but no signal was reported in the tissues of control group. In the malignant cervical tumors group, 43.6% has strong score (score III), whereas 37.5% and 18.8% has moderate score (score II) and low score (score I) respectively. While, in benign cervical tumor group, 57.1% were found tohave moderate score (score II) , 28.6% and 14.3% have strong score (score III) and low score (score I) respectively. - P16 protein overexpression was detected by IHC test in 63% of malignant cervical tumors, 56.3% of benign cervical tumors while none of positive result was reported in the tissues of control group. - The highest positive result for IHC of Rb and P16 according to cancer type was found in squamous cell carcinoma 61.1% and 72.2%, respectively, while there percentage in adenocarcinoma was 55.6% and 44.5%, respectively. - The highest percentage of Rb protein expression was found within poorly differentiated that have 100% (3 out of 3 cases) .Followed by 70% (7 out of 10 cases) and 42.9 % (6 out of 14 cases) within moderately and well differentiated respectively. - The highest percentage was 100 % (3 out of 3 cases), 64.3%(9 out of 14 cases), 50% (5 out of 10 cases) of cervical cancer tissues that showed IHC reaction for P16 protein have poorly, well and moderately differentiated respectively. - Among 22 cervical cancer tissues that showed infection with high oncogenic risk - HPV31/33 genotypes 100% and 77.3% have additional impact by P16 and Rb protein expression respectively. - Immunohistochemical examination of cervical tumor tissues for CD8+,CD16+ and CD20+ cell surface markers, revealed that CD8+cells the percentage of detection was 66.7% in cervical cancer group, 62.5% in benign cervical tumor group and 23.1% in control group. Also, CD16+ was detected in 29.6% of cervical cancer group, 31.3% of benign cervical tumor group and in 15.4% of control group. The CD20+ cells were detected in 44.4 % of cancer group, 43.8% of benign tumor group, and in 15.4 % of control group. - Among 22 cervical cancer tissues that showed infection with high oncogenic risk - HPV31/33 genotypes 77.3% have additional impact by CD8+ protein expression. Whereas 45.5%, 54.5% have additional impact of CD16+, CD20+ protein expression respectively
