The Characteristic Of NGF Expression In Glioma And Its Clinical Significance

Objective:To explore nerve growth factor (NGF) nuclear expression in astrocytoma and its biological meaning. Employ NGF as a carrier to deliver the Auger electron emitter iodine-125 to the nuclei of glioma cells for targeted radiotherapy.Methods:(1) 94 paraffin-embedded astrocytoma specimens were subjected to immunohistochemical (IHC) and hemotoxylin & eosin (HE) staining, preoperative cerebrospinal fluid (CSF) specimens and intraoperative snap-frozen astrocytoma tissue were assayed for NGF expression by ELISA and Western Blotting. The prognosis of corresponding patients was tracked.(2) To observe whether NGF nuclear expression was specific in astrocytoma, 56 cases of meningioma sections were immunohistochemical stained with NGF, PR, Ki67 antibodies. Corresponding patients were followed up for recurrence of meningioma.(3) NGF was radioiodinated with the Auger electron emitter 125I by Bolton-Hunter method. Absorbance values of U251 treated with 18.5,37,74, 148,296,592,1184,1480,1850,2590,3330,3700 kBq/ml 125I-NGF were detected by MTT assay. The corresponding minimum concentration of 125I-NGF tends to the minimum absorbance value was selected as minimum effective concentration, and was adopted in follow-up experiments. Plate colony-forming assay was adopted to compare the proliferation ability of cells treated with complete culture medium, 592kBq/ml Na125I, 1μg/ml NGF and 592kBq/ml 125I-NGF. Distribution of silver particles within the cells was observed by autoradiography to represent the existence of 125I-NGF inside the cells. DNA damage was evaluated by comet assay and micronuclei forming assay. The proportion of cells at G0/G1 or S phase were detected by flow cytometry.Results:(1) NGF-positive immunoreactive products were distributed in both cytoplasm and nucleus of astrocytoma, but were only located in the cytoplasm of traumatic brain injury (TBI) tissue. NGF nuclear-positive rate (NPR) of astrocytomas gradesⅢ-Ⅳ(70.0%) was higher than that of astrocytoma gradesⅠ-Ⅱ(28.6%, P<0.05). NGF-NP expression positively correlated with NGF concentration in CSF (r=0.755, P<0.01). Kaplan-Meier survival analysis indicated that the median survival time was 25 months in NGF-NP astrocytoma gradeⅠ-Ⅱpatients and 42 months in NGF nuclear negative (NGF-NN) astrocytoma gradeⅠ-Ⅱpatients (P<0.05). And in astrocytomaⅢ-Ⅳ, the value were 7 and 24 months respectively (P<0.01). Two types of NGF with molecular weights of 13 and 36 kDa were present in astrocytoma, but only 36 kDa NGF was found in the CSF. NGF expression elevated as the maligancy increased.(2) NGF nuclear expression also exist in meningioma. NGF nuclear positive rate (41.1%) is higher than Ki67 positive rate (35.7%) (P>0.05). PR positive rate was 60.7%. NGF nuclear positive rate positively correlated with Ki67 positive rate (r=0.741, P<0.01), while negatively correlated with PR-positive rate (r=-0.59, P<0.01). The median PFS time of NGF nuclear-positive patients is 36±2.484 months, which was significantly shorter than that of NGF nuclear-negative group (59±2.295 months) (P <0 .01).(3) Minimal effective concentration of 125I-NGF is 592kBq/ml. Under this concentration,125I-NGF could be transported to the nuclei of U251 and causes DNA damage. Being treated with 125I-NGF, U251 clone forming rate was (0.02±0.01), compared to control group (0.33±0.02), the proliferation of U251 was inhibited remarkably (P<0.01). The ratios of cells at S phase (10.69±0.02) decreased remarkably (P<0.01).Conclusions:(1) Although NGF nuclear expression has no specificity in astrocytoma, that is meningioma may also display NGF-NP staining, NGF-NPR positively correlated to Ki67 positive rate. The survival analysis indicated that NGF-NP expression was an indicator of tumor cells proliferation. NGF-NP expression and NGF level in CSF were significant prognostic factors in astrocytoma patients. Because of the easy access of CSF, combining with image diagnosis, NGF expression level in CSF may be developed as an indicator for the surveillance of astrocytoma, predicting the patients'outcome and evaluating treatment effctiveness.(2) After being iodinated with 125I, 125I-NGF can be transported to nuclei and causes DNA damage in U251. 125I-NGF has specific lethal effect to U251 cells at S phase.