Application Of Human Papillomavirus DNA Detection In The Prognosis Of Pelvic Lymph Nodes Metastasis In Cervical Cancer

BACKGROUDSCervical cancer is the second most prevalent malignant tumor among womenworldwidely. Infection with several so-called high-risk human papillomavirus (HPV)types is considered as a prerequisite for genesis and progress of the disease. Themechanism of HPV-induced oncogenesis has preliminarily been elucidated throughconsiderable details. Partial integration of viral genome segments into the host DNA,which may be consistent in HPV-infected ceils, results in the overexpression of E6and E7 gene, eventually leading to uncontrolled proliferation and immortalization. 15types of HPV have been confirmed to be associated with cervical cancer. Amongthem, type 16, 18, 45 and 31 are the most common ones. Prevalence of different typesof HPV varies geographically. Therefore, in order to enhance the efficiency ofscreening, the most common local HPV variants should be identified according todifferent regions.Recent research on prognostic variables in advanced cervical cancer indicatesthat not only are primary tumor histopathological parameters important, but also thepathological variables for pelvic lymph node metastases status. Metastasis in thepelvic lymph nodes was indicated as the most significant predictor of poor survival. Otherwise, even some early clinical staging, non-enlarged, non-metastatic lymphnodes cancers sometimes show worse outcome than would be expected. It indicatesthat there were some micromatastasis which could not detected with routinehistopathological examination. Hence, it would be useful to have more reliableparameters, which should allow a better identification of patients at high risk oflymph node metastases, local recurrences, and death. Some researcher has found thatHPV DNA can be detected in cancer free pelvic lymph nodes. It is worth to be payclose attention to the relationship between detection of HPV DNA in pelvic lymphnodes and metastasis of patients with cervical carcinoma.Moreover, based on the identification of prognostic factors and precise stagingof the disease, treatment of cervical cancer has evolved from standard radicalresections to more individualized approaches. The modality and circumsciption ofoperation for patients with cervical cancer are the controversial focal point for thepast many years. The incidence of positive nodes in the most favorable group ofpatients, stage IB1, is approximately 15%. This means that more than 80% of thesepatients who undergo lymphadenectomy derive no benefit from the procedure yetmust endure the associated increase in operative time, blood loss, risk of lymphocyst,and lymphedema. Thus, accurate determination of nodal status in patients with cervixcancer would allow triage of node-negative patients to receipt radical pelvic surgerywithout lymphadenectomy.Part 1 Associations of high-risk HPV types with clinicopathologicalcharacteristics of cervical cancerOBJECTIVE1. To study the prevalence of main high-risk HPV genotypes in primary tumors ofpatients with cervical cancer in Guangdong Province; 2. Analyze the relationship between the detection rates of high-risk HPV genotypesand the clinicopathological characteristics of patients with cervical cancer;3. To check the effects of HPV DNA detection in formalin-fixed, paraffin-embeddedcervical cancer tissues, compared to instant freezing specimens.METHODSThe investigated group comprised 55 patients suffering from cervical cancer(clinical stageâ… A toâ…¡B), which all visited the Department of Obstetrics andGynaecology, Nangfang Hospital, Southern Medical University, Guangdong, sinceNov. 2005 to Dec. 2006. They were all born and grow up in Guangdong Province.HPV L1 gene fragment in cervical cancer tissue was amplified by HPVospecific PCRwith general consensus primers GP5+/GP6+. The types of HPV were identified byPCR product sequencing. Thereafter, the correlation between high-risk HPV type andthe clinico-pathological features of cervical cancer was evaluated. In addition,comparison of the detection results between fresh freezing specimens andformalin-fixed, paraffin-embedded tissues which were obtained from 25 patients withcervical cancer was also performed.RESULTS1. The prevalence of high-risk HPV was 80% in these 55 patients with cervicalcancer. The most common HPV DNA type found here was HPV16, whichaccounts for 69.9%. Meanwhile, a rare HPV type in Guangdong Province, HPV45,was detected in one case.2. Presence of HPV18 and a multiple HPV infection of HPV16 and HPV18 weremore common in patients of stagesâ…¡, moderate to poor differentiation orhistopathologically confirmed metastasis than that of a single HPV16infection.3. There was no significant difference between the presence of HPV DNA in two types of the specimens, fresh freezing specimens and formalin-fixed,paraffin-embedded tissues. The degree of concordance for the two types ofspecimens was fine.CONCLUSION1. HPV16 is the most common HPV DNA type found in patients with cervicalcancer in Guangdong Province.2. HPV18 and the multiple HPV infection of HPV16 and HPV18 related to laterclinical stage, poor differentiation and metastasis of pelvic lymph nodesconfirmed by histopathological examination. Patients infected HPV18 and themultiple HPV infection can increase the clinical risk compared to HPV16infected.3. Selecting suitable primers can warrant the efficacy of detecting HPV DNA informalin-fixed, paraffin-embedded tissues of cervical cancer.Part 2 Comparison of two different methods for human papillomavirus detectionin paraffin-embedded cervical neoplastic lesionsOBJECTIVE1. Evaluate different molecular methods for HPV DNA testing in paraffin-embeddedtissue of cervical cancer.2. To find a better method for detecting HPV DNA.METHODSThe investigated group comprised 36 patients suffering from cervical cancer(clinical stageâ… A toâ…¡B), which all visited the Department of Obstetrics andGynaecology, Nangfang Hospital, Southern Medical University, Guangdong, sinceJan. 2005 to Dec. 2006. Two molecular methods for HPV DNA testing inparaffin-embedded tissue with cervical cancer were evaluated: in situ hybridization(ISH) and polymerase chain reaction assay (PCR) with sequencing.RESULTS1. The positive detection rate of HPV DNA in paraffin-embedded tissue of cervicalcancer were 77.8% and 24.5%, respectively for PCR and ISH.2. Positive samples detected by ISH displayed the same results when detected byPCR, while negative samples detected by PCR showed the same results whendetected by ISH.3. A rare type of HPV45 which cannot be detected by ISH, was detected by PCRwith sequencing.CONCLUSIONFor detecting HPV-DNA in paraffin-embedded tissue of cervical cancer, PCR isbetter than ISH.Part 3 Analysis of correlation between HPV DNA lymph node presence andmetastasis confirmed with histopathological examinationOBJECTIVETo investigate the correlation between HPV DNA lymph node presence andmetastasis confirmed with histopathological examination.METHODSThe investigated group comprised 31 patients suffering from cervical cancer(clinical stageâ… A toâ…¡A), which all visited the Department of Obstetrics andGynaecology, Nangfang Hospital, Southern Medical University, Guangdong, sinceJan. 2005 to Dec. 2006. HPV L1 gene fragment in paraffin-embedded primary tumorand pelvis lymph nodes was amplified by HPV-specific PCR with general consensusprimers GP5+/GP6+. Type of HPV was identified by PCR product sequencing. Thecorrelation between HPV DNA lymph node presence and clinico-pathologic parameters of primary tumour was then analyzed in cervical carcinoma patients.RESULTS1. The positive rate of HPV-DNA detection in the pelvis lymph node from 31 casesof cervical cancer was 58.1%.2. The positive rate of HPV-DNA detection was 84.6% in 13 cases withpathologically confirmed metastases. For the two cases in which HPV DNA wasnot found in corresponding metastatic pelvis lymph nodes, detection of HPV DNAwere also negative in the primary tumor.3. Presence in pelvic lymph nodes of the other 18 cases, which has no metastasesalready excluded by pathologically examination, is 27.8%, which is significantlylow than the metastatic cases.4. The risk factor of the presence of HPV DNA in the lymph node was similar to thatof histological metastases.5. Both result of HPV DNA detection and pathological examination indicate thatpositive samples were more detectable in obturator, internal iliac and externaliliac lymph node groups, which accounts for more than 90%.CONCLUSION1. HPV-DNA detection in the pelvis lymph node is a helpful prognostic factor ofmetastases.2. Obturator, internal iliac and external iliac lymph node groups are the mainsentinel lymph nodes.SUMMARY1. HPV16 is the most common HPV type found in patients with cervical cancer inGuangdong Province.2. Patients infected with HPV18 and the multiple HPV infection could have a increased clinical risk comparing to HPV 16 infection.3. Selecting suitable primers could warrant the efficacy of detecting HPV DNA informalin-fixed, paraffin-embedded tissue of cervical cancer.4. For detecting HPV-DNA in paraffin-embedded tissue of cervical cancer, PCR isbetter than ISH.5. HPV-DNA detection in the pelvis lymph node is a helpful prognostic factor ofmetastases.6. Obturator, internal iliac and external iliac lymph node groups are the mainsentinel lymph nodes.