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DISCUSSION
Retinoblastoma is the most common intraocular malignancy in infant and children, which can lead to the blindness, or even death. Currently, at clinic, most Rb have entered intermediate stage, or even terminal stage when it is diagnosed. For these Rb patients, enucleation is still the main therapeutic approach, which results in disfigurement of face and physical disability, and the cure rate is relative low[78]. In addition, the differential diagnosis between some atypical Rb, especially ectogenous Rb, and Coats disease is very difficult, because these two diseases represent leukocoria[3,9]. If retinoblastoma was misdiagnosed as other disease, it would be threatening childrens life on account of the delayed treatment; while if other diseases were misdiagnosed as retinoblastoma, the patients would be wrongly performed enucleation, which would lead to lifetime disability. So it is an urgent task for an ophthalmologist to make an earlier correct diagnosis and differential diagnosis of retinoblastoma.Table 1 Differential proteins in sera of Rb patients screened by IMAC30 chip(略)Table 2 Differential proteins in sera of Rb patients screened by CM10 chip (略)
Regarding the molecular targets of Rb, some scholars had studied the relationship between lactate dehydrogenase, neuron specific enolase(NSE) and retinoblastoma in serum and aqueous humor. Hervas et al[1] measured the content of NSE in sera from 17 retinoblastoma patients and 8 healthy adults by radioimmunity method and found that serum NSE level of 88% retinoblastoma patients was obviously higher than that of healthy adults. Wu et al[2] measured the content of NSE in sera from 23 retinoblastoma patients and 25 healthy adults by electrophoretic technique and found that NSE was detected in 78.2% retinoblastoma patients. Moreover, NSE exists not only in tumor tissue and blood of retinoblastoma patients but also in nervous tissue like retina. Besides, some diseases such as trauma, inflammation, ischemia, hypoxia in nervous system, small cell lung cancer, neuroendocine tumor and so on, can also give rise to the ascent of NSE content in blood[1012]. So the value of NSE is limited in early diagnosis and differential diagnosis of retinoblastoma. Furthermore, lactate dehydrogenase has a lower specificity and it is difficult to be applied widely in clinic. And genetic diagnosis of retinoblastoma is only suitable for the patients with Rb genetic mutation in germinal cell. However, about 60% Rb patients are not genetic type.
In recent years, surfaceenhanced laser desorption/ionization timeofflight mass spectrometry (SELDITOFMS) and protein chip technique have been widely applied in the field of searching biomarkers for early cancer detection[1318]. The mechanism of this technology is laser desorption/ ionization timeofflight mass spectrometry and protein chips made of a variety of chromatographic surfaces were used to selectively retain polypeptides and proteins over a wide range of relative molecular mass from 1kDa to 500kDa from crude extracts. It is of a rapid, simple, sensitive and highthroughput character and can detect many samples simultaneously. It can be applied directly to detect biological specimen, such as urine, blood, cerebrospinal fluid, cell disruption fluid and all kinds of secretion. Adam and his associates[13] reported promising results with sensitivity of 83% and specificity of 97% using the SELDITOF pattern for serum biomarkers of prostate cancer detection. Using the same technology in prostate cancer, Pan et al[19] found 18 marker proteins. Of the 18 proteins, four proteins of 15265，15868，16003，16068 m/z had high expression and the other 14 proteins had low expression. Based on the results, the classification tree for diagnosis of prostate cancer can distinguish 96.386% patients and 92.632% normal persons. Also, Kanmura et al[20] found 6 marker proteins with sensitivity of 83% and specificity of 76% when they made a study about early diagnosis of hepatocellular carcinoma(HCC) using the SELDITOF pattern. These studies demonstrated that the SELDITOF pattern had a broad clinical application prospect in the diagnosis of tumor, but its reproducibility and reliability need the standardization of detecting parameter and further clinical verification[21]. Yet, the proteomic approach has not been used to identify the protein markers of retinoblastoma at present. Our study chose two different protein chips which had different chemical modification surface in order to increase the number of captured proteins and provide valuable information about physicochemical property for marker proteins. IMAC30 chip can capture phosphorylated protein or/and metalbound proteins while CM10 can capture mainly hydrophilic proteins with high isoelectric point. Our results showed that IMAC30 chip captured 26 differential proteins while CM10 chip captured 4 differential proteins. Among them, two proteins, 5888m/z and 8590m/z, were captured by the two different protein chips simultaneously, which demonstrated that some differential proteins had many different physicochemical properties and were captured by different chips because of its structural difference. Therefore using different chips can make a supplementary effect and capture more differential proteins. Our study showed that there were rich phosphorylated and metalbound differential proteins in sera of Rb patients.
Using the SELDITOF pattern, we detected serum samples from 18 Rb patients and 17 agematched normal children and found that, among 28 differential protein peaks obtained from IMAC30 chip and CM10 chip, 23 peaks were expressed highly and 5 peaks were expressed lowly. We respectively used the differential protein 7014m/z and 7798m/z to carry out a statistical test, and results showed that the former had a specificity of 82.4% and a sensitivity of 94.4% and the latter had a specificity of 70.6% and a sensitivity of 83.3%. These results hinted that there were specific marker proteins in sera of Rb patients and SELDITOFMS and protein chip technique was possible to become a new important method for screening, early diagnosis and differential diagnosis of retinoblastoma. However, we still need identify what on earth the differential proteins are by some other methods and increase the number of samples to further confirm its validity and reliability in clinical application.
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