Detection Of High-risk HPV DNA And CK19 In Pelvic Lymph Nodes In Stage Ⅰa～Ⅱa Cervical Cancer And Its Significance

PrefaceIt has been suggested in a great deal of recent studies and practical results that the cervical cancers which severely threat women's health are the only kind of human malignancies whose incidence rate and motality rate can be reduced by medical interventions.One of the most important prognostic factors for cervical cancer is lymph node metastases.Studies note that patients with pathologically confirmed nodal metastases can reduce their postoperative recurrence rate as well as increase survival rate by receiving postoperative adjuvant radiotherapy or/and chemotherapy.Therefore, if it is possible to access the pelvic lymph node status precisely,then selecting individualized treatment strategy will be worth of serious discussion and exploration.Micrometastases refer to the scattered isolated cancer cells that can hardly be detected by common pathological examination.Along with the development of immunochemistry and molecular biology,it is now possible to detect single and insidious cancer cells that are concealed under HE stain in conventional pathology, providing new detection methods for studying lymph node micrometastases in cervical cancer.Cytokeratin(CK)is mainly used as a detection index in former studies on lymph node micrometastases in cervical cancer,yet detection rate of micrometastases in pathologically negative lymph nodes varies greatly by different methods:8-15%by immunohistochmistry,and 44%by polymerase chain reaction(PCR)with the micrometastases rate of 50%.Besides,HPV DNA which is integrated into cervical cancer cell nucleus is also applied for micrometastases detection,and by detection of HPV16 DNA using PCR the micrometastases rate of cervical cancer with pathologically negative lymph nodes is 26.3%-38%.We investigated detection methods of micrometastasis in lymph nodes in early-stage cervical cancer and its relation with the clinical pathological features,besides we additionally made a simple comparison between those two detection methods.Materials and MethodsResource of specimens and reagentsWe selected 28 paraffin embedded specimens of cervical cancer operated at China Medical University Affiliated Shengjing Hospital from 2003 to 2005.Based on the International Federation of Gynecology and Obstetrics(FIGO)staging criteria for cervical carcinoma,the patients included 15 cases of stageⅠand 13 cases of stageⅡ. Besides 26 cases showed squamous cell carcinoma and 2 adenocarcinoma.Pathological grading:7 cases were of high differentiation,14 of intermediate differentiation,and 7 of low differentiation.The mean age of the patients was 41 years(median:41 years, range:29-60 years).13 cases had lymph node metastases confirmed by pathological examination,and the other 15 did not.Paraffin embedded specimens were taken from the primary lesions and lymph nodes in the obturator and internal iliac regions of both sides.A total number of 104 lymph nodes were obtained from these 28 patients, averaging 3 to 4 ones per patient.According to the pathological metastatic condition, the lymph nodes were divided into three groups:group of metastatic lymph nodes from patients with lymph node metastases,group of non-metastatic lymph nodes from patients with lymph node metastases,and group of lymph nodes from patients without lymph node metastases. HPV DNA in situ hybridization DIG Staining System Kit(FISH 2004 HPV); CK19 monoclonal antibody,UltraSensiveS-P Immunohistochemistrical and DAB Coloration Kit。Preparation of sectionsThe paraffin embedded specimens of cervical cancer and lymph nodes were prepared for consecutive sectioning with thickness of 4μm.In situ hybridization and immunohistochemical method were performed respectively on those sections which had been definitely diagnosed by two experienced pathologists as cervical infiltrating carcinoma,metastatic lymph nodes,and non-metastatic lymph nodes.Detection of high-risk HPV DNA by in situ hybridizationHigh-risk HPV DNA probes were designed and synthesized(sequences:upstream 5'-CTGTG TAGGT GTTGA GGTAG GTCGT GGTC-3',downstream 5'-ATCAT CAACA TTTAC CAGCC CGACG AGC-3').This probes could be used in combination to hybridize successfully with positive template sequences of HPV types 16,18,31,33,35,39,45,51,52,56,58,59,68.The procedures were performed according to the kit direction.Assessment of results:positive hybridization signal was identified as brown yellow particles located in nucleus.Detection of CK19 using immunohistochemical methodCK19 was detected using immunohistochemical SP method according to the kit direction.Assessment of the result:yellow brown particles in the kytoplasm represented positive reaction.ResultsExpression of high risk HPV DNA in lymph nodes in three groups and their relation with the clinical pathological parameters of cervical cancer. All 28 primary lesions with cervical cancers displayed positive reaction when detecting the expression of high-risk HPV DNA using in situ hybridization(positivity rate,100%).From 13 patients with lymph node metastases,23 of 24 pathologically certified metastatic lymph nodes were high-risk HPV DNA positive(positivity rate, 95.8%).All high-risk HPV DNA positive lymph nodes were derived from 9(9/15) patients so that detection rate of micrometastases was 60%.Positivity rate of high-risk HPV DNA in all pathologically negative lymph nodes was 45%.The detection rate of high-risk HPV DNA in group of metastatic lymph nodes from patients with lymph node metastases was obviously higher than that either in group of non-metastatic lymph nodes from patients with nodal metastases,or in group of lymph nodes from patients without nodal metastases(P＜0.001).Detection rate of high-risk HPV DNA in lymph nodes in FIGO stageⅠpatients was 78.6%(10/14),and 85.7%(12/14)in stageⅡpatients,indicating that the detection rate of high-risk HPV DNA showed a upward trend along with increased stage(P＞0.05),a downward tendency with increased differentiation degree(P＞0.05),and no relation with patients' age(P＞0.05)Expression of CK19 in three lymph nodes groups and their relationship with the clinical pathological parameters of cervical cancer.All 28 primary lesions with cervical cancer revealed CK19 positive reaction.23 of 24 pathologically-confirmed metastatic lymph nodes from patients with lymph node metastases were positive for CK19(positivity rate,95.8%).Besides,of 57 lymph nodes from 15 patients without pathologically-confirmed lymph node metastases,14 showed CK19 positive(positivity rate,24.6%).As all 14 CK19 positive lymph nodes came from 7 patients,the detection rate of micrometastases was accordingly 46.7%(7/15). Detection rate of CK19 in all pathologically negative lymph nodes was 25%(20/80). Consistency test of the results of two detection methods.The consistency of the results of two detections methods—immunohistochemical method for detecting CK19 and in situ hybridization for detecting high-risk HPV DNA, was good.For 104 lymph nodes specimens,the detection rate by in situ hybridization was 56.7%(59/104),and 51%(53/104)by immunohistochemical method.The coincidence of two methods was 71.7%,and KI was 0.46.ConclusionsBoth detection of high-risk HPV DNA by in situ hybridization,and CK19 by immunohistochemical method could detect lymph node micrometastases in early-stage cervical cancer.As a method detecting on the molecular level,in situ hybridization was probably more sensitive for detection of lymph node micrometastases in early-stage cervical cancer.