| Backgroundprimary hyperparathyroidism (PHPT) is characterized by an increased secretion of parathyroid hormone (PTH) together with hypercalcemia caused by the increase of functional parathyroid tissue due to tumor or hyperplasia. With the medical development, this disease was increasing. Most patients in the developed world are now diagnosed on routine screening at an asymptomatic stage. Because of the clinical manifestation diversity and lack of understanding, the misdiagnostic condition of this disease often occurs, which resulted in patients suffering and high medical costs. Parathyroid cancer can cause the severe clinical symptoms and is an important cause of death in the group of patients with PHPT. However, parathyroid tumors are heterogeneous and the diagnosis of parathyroid tumor subtypes remains challenging. The histopathological diagnosis of parathyroid carcinoma can be a problem. Differentiation of parathyroid carcinoma from parathyroid adenoma especially atypical adenoma is very difficult in some cases Once diagnosed, parathyroid carcinoma is usually related with a high incidence of local recurrence and distant metastasis. On the basis of our hospital patients, we performed the gene expression profiles and molecular biology study of PHPT.Objective:1.To analyze the clinical characteristics of PHPT patients diagnosed in Peking Union Medical College Hospital from1975to2013. To investigate the change of PHPT clinical manifestation in different periods, age and pathological classification.2. To screen the differential genes between benign and malignant parathyroid tumor by gene expression profiles and validate these genes through modern molecular biology techniques. Then to find the genes used for the diagnosis of parathyroid cancer.3. To study the relevance between candidate genes and the clinicopathological features of patients in parathyroid cancer and provide the theoretical supports for the recurrence and prognosis of this disease.Methods:1. We conducted a retrospective study in Peking Union Medical College Hospital in China.490patients who were diagnosed with PHPT between January1975and June2010were enrolled in this study. All patients underwent operations and possessed complete medical datas. Medical records of patients were systematically reviewed, including demographic information, clinical and biological data, surgical and postsurgical data. The changes in different period, age and pathological classification were analyzed.2.We performed gene expression profiles study between normal parathyroid and parathyroid tumors including parathyroid hyperplasia, adenoma, atypical adenoma and cancer. Then the differential genes were analyzed through Go-analysis and Pathway-analysis. The candidate genes including CD24, HMOX1, VCAM1and KCNA3were selected through the constructed signal-net. Firstly mRNA level of these genes were examined by RT-PCR technique to evaluate the expression of CD24, HMOX1, VCAM1and KCNA3in normal parathyroid and parathyroid tumors tissue. Then immunohistochemical staining and western blotting were applied to evaluate these genes.3. Expression of CD24, HMOX1, VCAM1and KCNA3in parathyroid cancer tissues was investigated by immunohistochemistry staining; the association between CD24, HMOX1, VCAM1, KCNA3and clinical characteristics of parathyroid cancer was analyzed; the relationship between these genes and prognosis of parathyroid cancer was studied by statistical software.Result1. The results of first part(1) PHPT patents with surgical therapy was increasing and especially obviously after2010.(2) On the whole most patients are female (2.40:1). Compared patients over50years with that below50years, it is mostly seen in people over50years.(3) Compared patients before2010with that after2010, the time from onset of symptoms to diagnosis was obviously shorter.(4) Compared clinical manifestation before2010with that after2010, patients with mixed type was significantly decreasing, patients with renal and asymptomatic type were increasing.(5) Preoperative PTH level was positively correlated to preoperative serum calcium, ALP, serum creatinine and tumor size of patients, but was negatively correlated to postoperative serum calcium. Serum calcium was positively associated with ALP, serum creatinine, tumor size and postoperative serum calcium.(6) Compared with different pathological tumors, the results showed that PTH, serum calcium, serum creatinine and tumor size of patients in cancer group were higher than those in others groups although the age of patients in cancer group was more young.2. The results of second part(1) CD24, HMOX1, VCAM1and KCNA3genes were indentified according to gene expression profiles, go-analysis, pathway-analysis and signal-net.(2) RT-PCR experiment showed the following results. mRNA level of CD24was higher in parathyroid cancer and adenoma tissue than other tissue and especially higher in parathyroid cancer tissue. mRNA level of HMOX1and VCAM1were both higher in parathyroid cancer and atypical adenoma tissue than other tissue and HMOXlwas especially higher in parathyroid cancer tissue but VCAM1was higher in atypical adenoma. mRNA level of KCNA3was higher in parathyroid cancer tissue than other tissue.(3) Immunohistochemical staining confirmed the following results. CD24positive expression was located in nucleus and partly cytoplasm; CD24expression was.higher in parathyroid cancer tissue than other tissue. HMOX1positive expression was located in cytoplasm and partly nucleus; HMOX1expression was also.higher in parathyroid cancer tissue than other tissue. VCAM1positive expression was located in cytoplasm and partly nucleus; VCAM1expression was higher not only.in parathyroid cancer tissue but also in parathyroid hyperplasia tissue. KCNA3positive expression was located in nucleus; KCNA3expression was both higher.in parathyroid cancer and atypical adenoma.(3) Western blotting confirmed the following results. CD24expression was.significantly higher in parathyroid cancer tissue than other tissue. HMOX1expression was. higher in parathyroid atypical adenoma tissue than other tissue. VCAM1expression was both higher.in parathyroid cancer tissue and hyperplasia tissue, especially higher in parathyroid cancer tissue. KCNA3expression was higher.in parathyroid cancer tissue, atypical adenoma and hyperplasia tissue, but higher in atypical adenoma tissue.3. The results of third part(1) There was a relationship between high expression of CD24and the primary recurrence time after surgical operation. High expression of VCAM1was the recurrence times. High expression of HMOX1was associated not only with the primary recurrence time and recurrence times, but also with tumor size. But there was no relationship between high expression of KCNA3and clinicalpathological factors.(2) Kaplan-Meier analysis showed that expression of HMOX1was significantly associated with the overall and disease-free survival time of patients. It was also showed that expression of VCAM1was only related to the overall survival time of patients. Conclusion1. PHPT patents with surgical therapy was obviously increasing. On the whole most patients are female. It is mostly seen in people over50years.Compared patients before2010with that after2010, the time from onset of symptoms to diagnosis was obviously shorter, patients with mixed type was significantly decreasing and patients with renal and asymptomatic type were increasing. Compared with different pathological tumors, the results showed that PTH, serum calcium, serum creatinine and tumor size of patients in cancer group were higher than those in others groups although the age of patients in cancer group was more young.2. CD24, HMOX1, VCAM1and KCNA3genes were selected by gene expression profiles. Expression of CD24, HMOX1, VCAM1and KCNA3was higher in parathyroid cancer tissue than that in other parathyroid tissue. These genes might be useful for the diagnosis of parathyroid cancer and the differentiated diagnosis of parathyroid tumors.3. CD24, HMOX1, VCAM1and KCNA3can provide the theoretical basis for the recurrence and prognosis of parathyroid cancer. CD24, HMOX1, VCAM1and KCNA3may become a promising tool for early monitoring and therapy of parathyroid cancer. |
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