The Analysis Of Expression Of ERCC1, XPD Proteins And Quantitative Nuclear Grade On Prediction Of The Response To Platinum-based Chemotherapy In Advanced Epithelial Ovarian Cancers

一. STUDY BACKGROUND AND OBJECTIVEOvarian cancer is the leading cause of gynecological cancer death in China. The relatively asymptomatic nature of early-stage disease and the lack of adequate screening tests have resulted in the majority of cases presenting with stage III or stage IV disease. Standard treatment for advanced-stage disease involves cytoreductive surgery followed by the administration of platinum-based combination chemotherapy. Approximately 85% of patients will respond to this primary chemotherapy regimen. However, of those that initially respond to chemotherapy, the majority of patients will eventually relapse and die from chemoresistant disease. Understanding the factors that determine the chemoresistance of tumor cells is therefore an important area of investigation. DNA is thought to be the main target for the toxicity of platinum. The reactive complex binds to DNA to form inter- and intrastrand, DNA-protein cross-links, resulting in inhibition of both DNA replication and RNA transcription, arrest at the G2 phase of the cell cycle and /or programmed cell death. The survival of cells after exposure to platinum is governed by complex interations between a large number of proteins involved in DNA damage recognition,DNA repair, and the response of cells to unrepaired or misrepaired DNA damage.Recently, using multiple analytical approaches, we have identified at least five changes: 1) decrease drug accumulation;2) increased cellular glutathione;3) increased repair of platinum-induced DNA damage at the level of the whole genome and in specific DNA sequence;4) altered types of DNA lesions formed by platinum;and 5) altered amounts of platinum DNA damage required to kill resistant as compared to sensitive cells.One of the clearly important resistance mechanisms is the ability of resistant cells to repair platinum-induced DNA damage. The repair of platinum-DNA lesions is believed to occur primarily by the process of nucleotide excision repair (NER). DNA damage is recognized by the zinc-finger protein XPA in association with heterotrimeric replication protein RPA. The XPA-RPA complex then is believed to recruit the basal transcription factor TFIIH, a multisubunit protein that also plays a role in transcription, to the site of damage. Two subunits of TF IIH, XPB and XPD, have helicase activities that are believed to function in opening up the DNA around the adduct, thus allowing structure-specific nuclease to incise the DNA. The excision repair complementation group 1 (ERCC1) is key gene in the procession of the NER and the ERCC1-XPF heterodimer is responsible for cutting the strand on the 5' side of the damage^2'.Excision repair involves recognition of DNA damage, incision of DNA on either side of the lesion, generation of a new DNA strand to replace the excised segment, ligation of the new segment to the exsiting strand of DNA, and restoration of the normal chromatin conformation. Platinum-DNA adducts make the cells arrest at G2 of the cell cycles;on the contrary, XPD and ERCC1 let this procession continue as show the above. Based on this fact, we studied whether there are the differences of protein expression levels of XPD and ERCC1 between the sensitivity and the resistance to platinum-based chemotherapy in the epithelial ovarian cancer.There are characteristic differences in the nuclear architectures of cancer cells, compared with normal cells, and some anticancer treatments restore normal nuclear structure and function. Recently, advances in understanding nuclear structure have revealed insights into the process of malignant trasformation and provided a basis for the development of new diagnostic tools and therapeutics. Some authors' study showed quantitative nuclear grade may be predictors of long-term survival in patients with advanced ovarian cancer treated with cisplatint3l Therefore, the study was to evaluate correlations among clinical, pathological, nuclear morphometric and chemotherapy response variables and their prognostic value in advanced ovarian cancer.H. MATERIALS AND METHODSPatients. 78 continuous patients who consisted of serous ovarian cancer and clear cell cancer were included. These patients underwent surgery from 1999 to 2004 at the Women Hospital, Zhejiang University, suffering from FIGO stage HI or IV disease of primary epithelial ovarian cancer;furthermore, each patient was treated by platinum-based chemotherapy for =^6 courses.All of patients were divided into three categories: 11) 36 patients who presented sensitivity to platinum-based chemotherapy and 27 resistance to platinum-based chemotherapy in all of serous ovarian cancers;2) 63 patients with serous ovarian cancer and 15 with clear cell cancer;3) 13 patients with serous ovarian cancers treated by neoadjuvant chemotherapy.Tissue Specimens. Ovarian tissues prospectively collected by the pathologist were obtained from the department of pathology of Women Hospital, Zhejiang University. Tissue blocks of all patients from the formalin-fixed, paraffin-embedded ovarian cancer specimens were cut in approximately 5 u m sections, fixed on glass slides, and shipped to the authors' laboratory for furtherprocessing. Slides were individeually immunostained and Fuelgen's stained, analyzed by using a blinded coding system such that staining procedures and moicroscopic assessments were performed without knowledge of the histopathological diagnosis and response to primary chemotherapy.Discriminance criteria. According to WHO criteria (1979), complete response was defined as complete eradication of all evaluable disease, confirmed by perineoscopy. Partial response was defined as a > 50% reduction in the measurable lesions lasting at least 1 month. Progressive disease was a > 25% increase in the sum of the products of perpendicular diameters of all measurable lesions or the appearance of new lesions. Stable disease included those clinical circumstances that did not fit the definitions of objective response or progression. Progressive disease and stable disease patients were included in the non-responder category. Complete response and partial response were included in the responder category. Platinum sensitivity was defined by response to first-line platinum-based therapy (responder category ) or a progression-free interval of >6 months off treatment. Patients who progressed during treatment, had stable disease in response to initial platinum-based therapy, or relapse within 6 months were considered to have platinum-resistance disease. Criteria for persistence or recurrence included clinical evidence of disease (e.g., physical examination findings or measurable disease on computed tomography or magnetic resonance imaging or B ultrasonic) or a sustained rise in serum CA-125[4].Antibody Preparation. The anti-ERCCl monoclonal antibody was produced using BALB/C mice injected with fiill length recombinant human ERCC1 protein, and was obtained from DBS Biotechnology (Pleasanton, CA). TFIIH p80(H-150) (XPD) is a rabbit polyclonal antibody raised against a recombinant protein corresponding to amino acids 611-760 mapping at the carboxy terminus of TFIIH p80 of human origin, was obtained from Santa Cruz Biotechnology, Inc. Tissuesdetermined previously to express ERCC1 and XPD were used as positive controls. The ERCC1 and XPD protein were also expressed in the nuclear of the tumor cells. Negative expression was defined as no detectable nuclear staining in tumor cells.Imntunohistochemistry. Immunohistochemical staining of ERCC1 and XPD protein expression were performed on paraffin-embedded tissue sections. Only the primary ovarian cancer was sampled in each case and the expression was not investigated in the metastases. Sections with 5-micrometer thick were dewaxed and rehydrated using xylene and alcohol. Endogenous peroxidase was blocked by dipping the sections in 3% aqueous H2O2 for 10 min and antigen retrieval was performed with a 2 min boiling of high temperature of 110 °C treatment in 10 mM citrate buffer, pH 6.00. Following antigen retrieval, sections were incubated individually overnight at 4 °C with a mouse monoclonal antibody to the ERCC1 protein (1:50 dilution) or a rabbit polyclonal antibody to the XPD protein (1:50 dilution), and lightly counterstained with haematoxylin. Immunostaining was performed using the avidin-biotin peroxidase complex technique, using diaminobenzidine as a chromogen.Sections were analyzed using light microscopy by the same gynecologic pathologist for the percentage of nuclei staining positve for ERCC1 or XPD and the intensity of staining on a 0-3+ scale.The pathologist was blinded to clinical data. Any appreciable brown staining was considered positive and graded as follows: -, almost no staining of tumor cell nuclei;l+,barely detectable staining;2+,easily seen fine gradules were present diffusely throughout the nucleus;3+,staining was so strong that nuclear detail was obscured. The score of each section was derived from H-Score calculation[5].Fuelgen's staining and analysis Sections were stained through Fuelgen's staining and analysed with a computer-assisted system (HPIAS-1000).Statistical analysis. Results were expressed as mean+SD and statisticalanalysis is processed in SPSS 11.0. Paired samples test, t test, Row Column Chi-square test or One way ANOVA were used to perform two or more groups difference analysis respectively. Spearman correlation analysis was used to test two groups correlations. The statistical analysis tests were two-sided, P<0.05 is regarded as significant difference.H. RESULTSERCC1 and XPD proteins overexpressed in the chemoresistant of serous ovarian cancer. Mean values for ERCC1 and for XPD in the resistance for platinum-based chemotherapy of serous ovarian cancer were approximately 50% and 40% higher than those seen in the sensitivity for platinum-based chemotherapy of serous ovarian cancer .however, their mean ages were not significantly different.ERCC1 and XPD proteins expression were not different between sensitivity and resistance for platinum-based chemotherapy in the clear cell tumors. Slides from fifteen patients suffered from clear cell tumors were individed into sensitivity group (eight patients) and resistance group ( seven patients) .All patients had FIGO stage III or IV disease. The scores of nuclei with positive ERCC1 and XPD immunostaining in the sensitivity versus resistance to platinum-based chemotherapy of clear cell tumors were not significantly difference.ERCC1 and XPD proteins expression of serous ovarian cancer were lower than those of clear cell tumors. For ERCC1, serous ovarian cancer had a little lower levels protein expression with P equal to 0.04. For XPD, clear cell tumors had approximately 30% higher than serous ovarian cancer with P<0.01.ERCC1 and XPD protein expression increased after cisplatin chemotherapy. Neoadjuvant chemotherapy had an effect on the expression of ERCC1 and XPD protein in the serous ovarian cancer. After cisplatin treatment immunohistochemical expression showed 23% and 32% higher than before.Coordinate expression of ERCC1 and XPD in the epithelial ovarian cancer. This relationship approximated a straight line with a r2 of 0.414, and the equation was ERCCl=0.573XPD+0.945. The data show that under baseline conditions,each 1.0 unit increase in XPD protein expression was associated with a 0.573 unit increase in ERCC1 protein expression. This suggested coordinate expression of these two genes.Mean nuclear area and perimeter of resistance to platinum chemotherapy were larger in the serous ovarian cancer,compared with sensitivity. Mean nuclear area and perimeter of serous ovarian cancer for chemoresistant disease were larger than sensitivity disease individually with P=0.003 and P=0.009.m. CONCLUSIONS1. Expression of ERCC1 and XPD proteins in clear cell carcinomas were higher than in serous ovarian cancers and those of resistance to platinum-based chemotherapy were higher than sensitivity in serous ovarian cancer. Platinum could induce an increase of NER-related genes including ERCC1 and XPD. The results suggested ERCC1 and XPD proteins were involved in resistance process for platinum-based chemotherapy in epithelial ovarian cancer.2.Quantitative nuclear image parameters, expression of ERCC1 protein were correlated with the response of platinum-based chemotherapy, which revealed some nuclear functions in some measure.3.ERCC1 protein expression and mean nuclear area were good predictors of the response of platinum-based chemotherapy in advanced serous ovarian cancer.