| BACKGROUNDThe breast cancer is one of the most popular malignant tumor of female and it stage the first for its mobidity and motality rate in western country. In recent decades, the motality rate in our country is steadily increasing and more and more young patients suffered it which severely damage the female’s health.In the past decades, there has been great improvement in the therapeutic effect of breast cancer. For example, neoadjuvant chemotherapy, combined radiotherapy, endocrine therapy and immuno-therapy had decreased the recurrence rate, and increased survival rate and life quality. While it is still unsatisfied for the therapeutic effect of cancer especially for late stage of breast cancer. So it is valuable to find a method to discover the breast cancer in the early stage. We should focus on the discovery of precancerous lesion in probing the early cancer. It is effective to dectect the precancerous lesion and make a microdissection to removal of the lesion. In the physical examination center, the breast mass can be detected by ultosonography and mammography and distinguished by pathology. While it can not be pointed out the appreciation rate. So it still should be judged by combinating the tumor amplication factors.It has been repeatedly researched the factors correlated the prognosis of breast cancer and it is still the focus nowdays. It is generally considered that the prognosis of breast cancer is assotiated with the clinical stagement, pathological stagement, histological form, P53gene, C-erbB-2gene, steroid hormone receptor, tumor amplification fraction et al. And more and more factors correlating the prognosis have been putforwarded, such as epiphilial growth factor receptor, nm23gene, E-calcium glutinous protein et al. What interested us is that detecting the concentration of this kind of abnormal proteins in serum will help us to evaluate the occurance of malignant tumor without invasive examination. The theory of tumor cell amplifying factors is based on the fact that the tumor cell lose normal regulating which lead to malignant proliferation. This theory guides to developing an accuracy and non-invasive serum detection method which could detect the potential malignant transformaiton earlier than equipments available. So it has been kept seeking a serologic and histological biomarker which correlats the amplification of tumor cell. In early1970, the discovery of CEA which is called’"embryonic tumor antigen" made people to believe that a new tumor specific antigen had been found. Nowdays a series of monoclonal antibody such as CEA, CA199, CA125, CA153still have controversial clinical use because of their specificity have missed our expectation. But the study via CEA and other embryonic antigen has provided us many valuable informations about circulatory tumor biomarker. Her2is a new practical protein biomarker of breast cancer which is appropriate for seriologic and histological detection and immno-therapy. While the expression of Her2gene in breast cancer is only25-30%which also resticts the applied range.Thymidine kinase (TK) is a rate-limiting enzyme in DNA salvage pathway which is also important biomarker evaluating the cell multiplification. There are two forms of isozymes in human cells which is TK1in cytoplasm and TK2in mitochondria respectively. TK1is called special enzyme in S stage which concentration is positively correlated to the synthesis of DNA and present periodic variation. The former study showed that the TK1in non-proliferation cell as well as serum and tissue of healthy people can hardly be detected. While in patients with malignant tumor, TK1increased along with the rapid proliferation of tumor cells. At present, there are many reports about the relationship between the hazard level of tumor and expression of TK1in lung cancer, cervical cancer and colon cancer et al which all considered that the expression of TK1was positive correlated to the hazard level and influence the prognosis. More study focused on the TK1concentration in serum and its changes in use of tumor screening, assisted diagnosis, curative effect survey and judgement of prognosis except for expression in the tissue.PART1Clinical significance of Thymidine1(TK1) expression in breast cancerObjective:To study the expression of TK1in patients with breast cancer, patiets with benign lesion of breast and healthy people and to study the difference of concentration in serum and their clinical significance.Method and ProcedureObject:(1) Choose the patients with unusual ductal hyperplasia (UDH), atypical ductal hyperplasia (ADH), ductal carcinoma in situ (DCIS) and invasive ductal cancer (IDC) to study the expression of TK1. Among of them, UDH (n=25), ADH (n=25), DCIS (n=25), IDC (n=60) and healthy breast tissue (n=20) as control came from the Pathology Department of ShenZhen2nd People Hospital2010-2012. The pathological stages of the specimens was in basis of American Joint Cancer Community (AJCC) standard and grading of World Health Organization standard(WHO). IDC grading:1n=12,2n=34,3n=14; stages:Ⅰ n=23, Ⅱa n=19, Ⅱb n=19, Ⅲa n=2. DCIS grading:1n=12,2n=11,3n=2; stages:all zero stages. All the precancerous lesion ADH were basis of WHO recommended standards.(2) The specimens of IDC were proceeded regular pathological examinations (HE stain) to make sure of their histological type, pathological characteristics (differentiation level and lymph node metastasis) and clinical stages. The immuno-histochemical method (MamVison) were used to detect the expression of ER, PR, p53, C-erbB-2. Ki-67and TK1of the tissue.(3) Collected60peripheral blood samples of breast cancer,20peripheral blood samples of benign breast tissues and10peripheral blood samples of healthy people. The concentrations of serum TK1in all the blood samples were measured by enhanched chemiluminesence (ECL) dot blot assay. All patients confirmed their enrolling in this experiment.Immuno-histochemical MaxVison method detect TK1expression in the tissue of breast cancer: Paraffin embedded specimens were continuously sliced to4micron in thickness. Sections were dewaxing with dimethylbenzene, gradient alcohol melt. PBS washed3min for3times. Antigen repair with microwave:Immerse the sections into dying jar with citric acid buffer solution (PH=6.0), heating to boiling with microwave,5-10min interval, total15-20min. The sections were washed by PBS3min X3times afer natural cooling. Each section was added with one drop of H2O2, incubation10min in room temerature to inactivate endogenous peroxidase. PBS wash3min×3times. Add antibody1, incubation in room temperature for60min. PBS wash3min×3times. Add antibody2, incubation in room temperature for30min. PBS wash3min×3times. DAB coloration:0.85ml distilled water, adding1drop of reagent from A, B, C box respectively, sufficiently blending, coloration keeping out of sun in room temperature3-5min, washing with distilled water. Haematoxylin slightly dye again, kohlrabiblue15min with tap-water, gradient alcohol dehydration, transparent with dimethylbenzene, sealing slices with neutral gum at last,observation with optical microscope.Enhanched chemiluminesence (ECL) dot blot assay:Prepareing antibody diluent, washing solution and sealant of PH=7.6with distilled water. Put calibrator1,2,3to nitrocellulose membrane A1, A2, and A3respectively with each dot3microlitre. Put the serum of inspetion pending into the empty dots in sequence with3microlitre per dot, natural cooling in room temperature for30min. Washing the membrane twice in the reaction box with configurated diluent for1min each. Adding sealant for30min. Adding TKl-1gY antibody with1:500dilution, jolt reaction for120min in room temperature. Washing the slices with cleaning mixture3times after abandoning the reactive solution,5min per time. Adding biotinylationized anti-IgY second antibody, reacting for40min in room temperature. Washing the slices with cleaning mixture3times after abandonning the reactive solution,5min per time. Adding ECL glow reagent, melting after passing through membrane, reacting accurately1min, and drying with absorbant paper, recounting for5min and crush remainder solution in the transparent film. Put the membrane into the chemiluminescence analysis meter, taking a picture and analysis after5min.Results(1) The positive stained of TKl in normal was negative or week positive. While in UDH. only quite a few cells were stained which located in the plasma. In the tissue from DCIS to IDC, the staining was deepened gradiently. The location was mainly in plasma, and some in nucleus.(2) During the60cases of breast cancer patients, the positive expression rate of TK1was85%(51/60). There were significant differences in histological grading,size of tumor, TNM stages (P<0.05), while not significant differences in age and metastasis of axillary lymph nodes.(3) TK1expressed at the cellular level, so the number of stained tumor cell could ensure us the expression of TK1in different tumor tissue. In the normal tissue, TK1did not express or less than5%, while in UDH increased significantly to3.3±1.4%, in ADH to8.6±2.8%, and there was significant diffenrence (P<0.001) between UDH and ADH.. In DCIS, the expression of TK1increased significantly to20±14%, while even higher in IDC to29.1±21.4%. In this experiment,80-90%of patients of ADH, DCIS and IDC are higher than5%, while in patients of UDH, only10%were higher than5%.(4) During the patients of DCIS and IDC, the expression of TK1increased gradiently as the stage from Ⅰ to Ⅲ. During the patients of IDC, although there were some overlap between different stages, the expression of TK1was positive correlated to stage. The mean of TK1was stage I and II a (P=0.029), stage Ⅰ and Ⅱb and Ⅲa (P<0.001), Ⅱa and Ⅱb (P=0.196). The expression greatly increased in patients with tumor size larger than3cm.(5) The average age of the patiets with ADH, DCIS and IDC was40.6±8.1,45.3±5.6,50.3±13years respectively, and the average age of UDH was43.5±7.7years.(6) There were significant differences between the healthy people, breast cancer patents and benign lesion patients in TK1expressed in peripheral blood (P<0..001). There were also significant differences between the breast cancer patients belonging to different stages (P<0..001). The concertration of TK1in peripheral blood increased along with the stage rised.Conclusions(1) There were differences of TK1expression between different histological grades and TNM stages and inceased along with the histological grade and TNM stage rised, which implied that TK1is a sensitive biomarker reflecting the proliferation of breast cancer.(2) The high positive expression of TK1and high pathological grade were both high risk factors for metastasis of breast cancer.(3) The concentration of TK1in peripheral blood inceased in sequence of healthy people, benign breast lesion, breast paitients. Serum TK1is an easy and helpful biomarker for diagnosis of breast cancer.PART2The clinical significance of serum TK1in breast cancer with neoadjuvant chemotherapyObjectives:(1) To discuss the concertration of serum TK1in local late-stage breast cancer with neoadjuvant chemotherapy.(2) To discuss the prognotic significance of serum TK1to breast cancer patients.Method and ProcedureObject:A total of48patients with LABC were recruited. None of these patients had inflammatory breast cancer. Treatment and surveillance protocols were standized to ensure study homogeneity. Compliance with treatment and surveillance protocol was95%and99%, respectively. All patients underwent standard treatment protocol for neoadjuvant chemotherapy. The majority of patients received four cycles of cyclophosphamide and epidriamycin, followed by four cycles of docitaxel. Surgical treatment consisted of either a modified readical mastectomy or breast conservation therapy(BCT, lumpectomy with tumor-free margin, axillary node dissection, and breast irradiation). Adjuvant axillary irradiation, systemic chemotherapy, and antiestrogen therapy were offered and administered as indicated per current standard of care. Surveillance protocol consisted of a history and physical examination every3months for first years, every6months for second year and annually thereafter. Annual chest x-ray, mammogram, complete blood count, and liver function test were obtained. Any additional radiological and/or histological evaluation was performed based on the patient’s examination and history. Clinical data were accrued and recorded prospectively and included age at diagnosis, comorbid conditions, stage of disease, treatment protocol, surveillance protocol compliance, and study endpoints. Study primary endpoints were cancer recurrence and cancer related death. Serum samples were taken from the patients in the following time, before neoadjuvant chemotherapy, after each cycle of neoadjuvant chemotherapy, after operation, after each cycle of adjuvant chemotherapy,3,6,12and24months post-adjuvant chemotherapy. For logistical reasons,48patients were analyzed at the time of before neoadjuvant chemotherapy, before operation, before adjuvant chemotherapy and3months after adjuvant chemotherapy.During the follow-up, a total of eleven patients developed distant recurrence, while seven patients developed loco-regional recurrence within3years of surgery. One patient developed distant recurrence after2years of surgery. The tumors were scored for the patient’s age, ER and nodal status. The blood sera were stored at-80℃Sera from15healthy individuals were used as negative controls. At the time of determination the sera thrawed and immediately assayed for S-TK1. Informed consent was obtained from all patients and the study was approved by the Committee on Research Ethic at Shenzhen University Hospital, China.Assay for S-TK1The concentration of S-TK1was measured by enhanced chemiluminescence (ECL) dot blot assay provided by Sino-Swed Molecular Bio-Medicine Research Institute,Shenzhen, P.R. China. Briefly,3ul of serum sample were applied to a nitrocellulose membrane, in duplicate. The sera were probed with and without anti-TK1chicken immunoglobinY (IgY) antibody, the latter were used as negative controls. Sera from13healthy individuals were also used as negative controls. We also used anti-TK1mouse immunoglobin G (IgG) monoclonal antibodies,with identical results. The ECL-treated membranes were exposed to X-ray films, taking into account the variation in S-TK1concentration of the samples. The intensities of the spots on the films were determined using a GS-700Imaging Densitometer(Bio-Rad, USA). The area of the spots were equally defined by intergration computer program of the GS-700Imaging Densitometer. From the three different concentrations of TK1, a standard curve was created, permitting calculation of S-TK1, as pmol/1(pM). The accuracy of the assay was4-6%. The sensitivity varied from0.75to1.0, depending on the type of malignancy, and the specificity was found to be1.0at a cut-off value of2pM. Figure1shows an example of the dot-blot and Westen blot analyses.Assay for HER-2expression A positive HER-2status is defined as two or more, using FISH method.Estrogen and progesterone receptor status Estrogen receptor (ER) and progesterone receptor (PR) status was determined using immunohistochemical methods. Activity greater than ten percent was considered positive.Results(1) Forty-eight patients were accured for this study. The mean age at diagnosis was49years, and the mean follow-up was28months. Due to the advanced nature of the disease, the majority of patients (46patients) underwent a modified radical mastectomy. There were22patients who developed recurrent disease, of which18patients (81.8%) had distant disease. The median DFS and OS were38months for each. Note that concentration of S-TK1was observed in varying degrees in breast cancer serum. Based on our previous study [20], We use2.0as our cut-off. Patients were distributed into two groups:(1) low S-TK1group (<2.0pM, n=19patients), and high S-TK1group (≥2.0pM, n=29patients).(2) The breakdown of patients as grouped by tumor size (T stage) and nodal status (N) following neoadjuvant chemotherapy is shown in Table1. The T stage distribution was as follows:TO lesions (n=3) T1lesions (n=5), T2lesions (n=24), T3lesions (n=14), and T4lesions (n=2). There were35node-positive patients and13node-negative patients. The N stage distribution was as follows:N0=13patients, N1=14patients, N2=12patients and N3=9patients.(3) To assess the robustness of our sample set, we evaluated outcome with known traditional prognostic markers. Among traditional clinicopathologic factors,nodal status was the strongest predictor of outcome;patients who had a high number of positive pathological nodes after neoadjuvant chemotherapy had a worse DFS(P=0.006) and OS (P=0.036) than those who had none or minimal nodal disease. These results indicate that our sample size was adequate.(4) Note that patients who had elevated S-TK1levels in their serum had a higher rate of recurrence and cancer-related death when compared to those who had low S-TK1levels. The5-year DFS for the low S-TK1group versus the high S-TK1group were56%and21%, respectively (P=0.003). The median DFS had not been reached for the low S-TKI group and was23months for the high S-TKl group. The median OS for the low S-TK1group and high S-TK1group has not been reached. There were not statistically significant differences between the two groups. Although high S-TK1concentration appeared to be the predictive of outcome, we wanted to know whether S-TK1was a covariant of nodal status. In other words, was high S-TK1levels a surrogate marker of advanced nodal disease? There was no statistical correlation between the two variables (.P=0.11), thus confirming that high S-TK1concentration was not a covariant of nodal status. Next, we evaluated to see whether high S-TK1concentration correlates with any known clinicopathological factors such as tumor size, nodal status, estrogen and progesterone receptors status, and HER-2receptor status. There were no correlations between the degree of S-TK1concentration with tumor size (P=0.15), nodal status (P=0.11), ER status (P=0.63), PR status (P=0.45). or HER-2status (P=0.89), thus suggesting that S-TK1concentration may be an independent predictor of outcome.(5) Finally, to further strengthen our hypothesis that high S-TK1concentration in serum following neoadjuvant chemotherapy is a noval independent prognostic indicator of poor cancer outcome in patients who have LABC, we performed a Cox Regression Analysis to compare the relative risks of cancer recurrence and cancer death between S-TK1and known clinicopathologica factors. Note that for both DFS and cancer-related death, S-TK1overexpression out-performed nodal status as a predictor of outcome. Patients whose serum had high S-TK1concentration following neoadjuvant chemotherapy had a higher risk of cancer recurrence compared with patients whose is low (P=0.003). In comparison, patients who had evidence of nodal disease had a higher risk of cancer recurrence compared with patients who had no evidence of nodal disease (P=0.002).Conclusion:(1) The breast cancer patients with high serum TK1concentration in peripheral blood have more cancer related mortality and recurrence risk than those with low TK1concentration.(2) S-TK1could be an independent factor for prognosis of local late breast cancer. |
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