| Objective Malignant gliomas are the most common primary brain tumor. Post-operative radiotherapy has been recommened as standard therapy for patients with malignant glioma. On routine follow-up MR images, the differentiation of recurrent tumor from radiation injury in subject previously resected and irradiated glioma is problematic, although both the lesions can be associated with more specific characteristics, such as the former with corpus callosum involvement or multiple enhancement. As a most widely used tracer,18F-flurdeoxyglucose positron emission tomography (18F-FDG PET) imaging is playing an increasingly important role in the diagnosis, grading, prognosis, response to the therapy, and differentiation recurrent from radiation injury of brain glioms. But FDG PET has some limits in clinical application. There are two parts in the study, including clinical and experimental researching of PET imaging in brain glioma and C6glioma model.(1)The aim of the study is to investigate the usefulness of FDG PET/CT for differentiation of brain glioma necrosis and recurrence after radiotherapy, in comparison with "C-methionine (MET) and "C-choline (CHO).(2)To determine the accuracy of multiple tracers PET in evaluating radiotherapy of rat C6glioma model.(3)The change of rat C6gliomas in the histopathology and molecular biology before and after radiotherapy was observed. To correlate the change with multiple radiotracers PET, using HE, Immunohistochemistry, RT-PCR and Western blot and test the affected mechanism of multiple radiotracers PET.Materials and Methods (1)From Sep.2005to Sep.2009, thirty-eight patients with suspected brain gliomas recurrence after radiotherapy referred to our hospital were examined with CHO, MET, and FDG PET/CT and find39lesions. Twenty-four patients accepted MET and FDG PET/CT, fourteen patients accepted CHO and FDG PET/CT. The diagnosis of28patients was confirmed by histopathology after surgery or biopsy, whereas that of the other11patients was made by imaging or clinical follow (>6months). PET results were evaluated by visual and semiquantitative analysis.①For visual analysis, the positive diagnosis was made when the FDG accumulation of lesions was more obvious than the adjacent or contralateral normal white matter. On the contrary, the negative diagnosis was made.②For semiquantitative analysis, the standardized uptake value (SUV) and tumor to contralateral normal white matter (T/N) ratio were calculated. The PET results were compared with histopathology or clinical diagnosis. The sensitivity, specificity and accuracy of FDG and MET, CHO PET/CT for differentiation of brain glioma necrosis and recurrence were calculated. The T/WM of brain glioma necrosis and recurrence on FDG and MET, CHO PET/CT were compared.(2)2.1The C6rat glioma cells were maintained in Dulbecco’s modified Eagle medium (DMEM). The cells were cultured monolayerly at37℃in a5%CO2and100%humidity incubator. For injection, the cells were harvested at phase of growing logarithmically, washed3times with phosphate-buffered saline, and counted cells.2.2One hundred and eighty-five male SD rats weighing180-200g and aged4-5weeks were used in this study. The rats were anesthetized with inhalation of aether. Then,1×108/ml cell suspensions were implanted subcutaneously into the right inguinal area.2.3Two weeks after C6cells implanted, it was considered successful that the diameter of nodules in the right inguinal subcutaneous area was larger than1cm.2.4Radiation treatment:6MV X-ray produced by Siemens Primus accelerator was used to treat SD rats with tumor. The dose rate was2Gy/min and standard SSD (source to skin distance, SSD) radiation was used. The total dose of single fraction irradiation was12Gy. The radiation fields cover the tumor adding1.5cm margin and0.5cm thickness tissue equivalent was used.2.5After C6glioma model successfully established, the rats were randomly classed into2groups:control group and radiotherapy group. The control group did not accept radiotherapy and was randomly classed into2groups:Bl, accepted PET/CT examination2weeks after C6cells implanted; B2, accepted PET/CT examination3weeks after C6cells implanted. The radiotherapy group was randomly classed into2groups:R1, accepted PET/CT examination24hours after radiotherapy; R2, accepted PET/CT examination1weeks after radiotherapy (3weeks after C6cells implanted). The every rat in all groups only accepted randomly one11C-tracer PET examination, and accepted FDG PET examination in the same day. 2.6PET was performed by use of a GE Discovery LS PET/CT scanner. The tail vein was used for administration of11C tracers and FDG11C tracers PET was performed5minutes after intravenously injection of0.2mCi radiotracers. FDG PET was performed2h after11C tracers’administration and1h after0.1mCi FDG administration. The data were transmitted to Xeleris workstation. CT, PET and PET-CT fusion images were obtained. The tumors SUVmax, size, and the SUVmean of contralateral muscle beside the spine were calculated. The T/M of tumors and muscle was calculated.(3)3.1The experimental animals and PET examination were identical to Part Two.3.2The animals were euthanized after examination. The tumors were extirpated by surgery and divided into two groups. One part was fixed with10%formalin and embedded in paraffin. The other was preserved in the liquid nitrogen at-70℃.3.3The sections were stained with hematoxylin and eosin to assess the cellular density. The sections were immunostained for Glucose transporter1(GLUT1), CD98, VEGF, HIF-la, p53,microvessel density (MVD), Ki67LI. C6gliomas apoptosis before and after radiotherapy was assessed using Terminal deoxynucleotidyl transferase-mediated dUTP nick-end-labeling (TUNEL).3.4The expression of4F2hc, LAT, VEGF, p53and HIF-la mRNA were tested using RT-PCR.3.5The expression of GLUT1, CD98, VEGF and p53protein was tested using Western blot.3.6The parameters mentioned above were compared using ANOVA and correlated with PET T/M.Results (1) There were28patients with recurrent tumors and11with radiation necrosis.①For visual analysis, the sensitivity, specificity and accuracy of FDG PET/CT for differentiation of brain glioma necrosis and recurrence were85.7%,81.8%and84.2%, respectively. MET PET/CT were94.4%,87.5%and96%, respectively. CHO PET/CT was90%,75%and86%, respectively.②For semiquantitative analysis, FDG_T/WM of brain glioma necrosis and recurrence were2.71±2.21,1.59±0.97,P=0.048; MET_T/WM of brain glioma necrosis and recurrence were4.25±1.88,1.48±0.81, P=0.001; CHO_T/WM of brain glioma necrosis and recurrence were15.33±13.35,7.44±1.77, P=0.038. The accuracy of combination FDG with MET or CHO PET/CT for differentiation of brain glioma necrosis and recurrence were96%and92.8%, respectively.(2)2.1Of the185rats,3did not have C6glioma, the rate of successful generation of a C6glioma was97.7%. But5rats died during radiotherapy or PET/CT examination.177rats were enrolled in this study at last.2.2The mean size of tumors of B1, R1, R2and B2group were8.61±6.57cm3,7.80±7.31cm3,5.21±5.48cm3and8.88±6.16cm3, respectively. The difference of tumors size between R2and R1was not statistically significant (P=0.556). There was statistical significance between B1and B2(P=0.014, P=0.008).2.3All rat C6gliomas accumulated radiotracers obviously but differently. The SUVmax of FDG, ACE, MET and CHO in B1group was10.81±5.65,3.63±0.75,.63±1.20and1.50±0.42. FDG SUVmax was higher than the other radiotracers (P=0.00). The T/M of FDG, ACE, MET and CHO in B1group was8.52±4.67,1.67±0.40,2.10±0.80and1.70±0.53. The FDG T/M higher than the other radiotracers (P=0.00).The SUVmax of FDG, ACE, MET and CHO in R1group was7.91±5.50,4.12±3.26,3.05±0.86and0.67±0.39.FDG SUVmax was higher than the other radiotracers (P<0.05). CHO SUVmax was lower than MET and ACE (P<0.05). The T/M of FDG, ACE, MET and CHO in R1group was7.98±4.38,1.47±0.33,1.59±0.38and1.75±0.73. The FDG T/M was higher than the other radiotracers(P=.00)。The SUVmax of FDG, ACE, MET and CHO in R2group was6.35±3.25,2.64±0.90,2.63±0.59and2.74±0.81. FDG SUVmax was higher than ACE, MET and CHO (P=0.00). The T/M of FDG, ACE, MET and CHO in R2group was5.06±3.21,1.32±0.34,1.40±0.32and2.36±0.93. FDG T/M was higher than ACE, MET and CHO (P=0.00). CHO T/M was higher than ACE and MET(P=0.01、P=0.00). The SUVmax of FDG, ACE, MET and CHO in B2group was10.05±3.20,2.64±0.68,2.86±0.76and2.14±0.51. FDG SUVmax was higher than the other radiotracers (P=0.00). The T/M of FDG, ACE, MET and CHO in B2group was8.17±4.28,1.62±0.34,1.61±0.44and1.81±0.54. The FDG T/M was higher than the other radiotracers (P=0.00). Among B1, R1, R2and B2, the FDG SUVmax and T/M of R2group were lower than B1and B2group (P<0.05). The CHO SUVmax of R1group was lower than the other3groups. The T/M of R2group was higher than the other3groups. The ACE SUVmax and T/M of R2was lower than B1and B2. The MET SUVmax of B1was higher than R2and B2, and the MET T/M of R2was lower than B1.2.4There was a positive correlation between FDG T/M and tumor size and cell density. No significant correlation was found between the other parameters and tumor size.(3)3.1In B1, R1, R2and B2groups, the GLUT1expression was0.231±0.1,0.149±0.07,0.026±0.01,0.209±0.19, respectively. The CD98expression was0.29±0.05,0.20±0.15,0.06±0.01,0.26±0.07, respectively. The4F2hcmRNA expression was2.93±0.12,2.27±0.21,0.26±0.05,2.58±0.28, respectively. The LAT mRNA expression was2.67±0.31,2.14±0.29,0.39±0.05,2.45±0.23, respectively. The HIF-1α mRNA expression was3.13±0.21,0.95±0.45,0.61±0.34,2.63±0.56, respectively. The VEGF mRNA expression was1.25±0.04,1.15±0.12,0.46±0.06,1.15±0.24, respectively. The VEGF expression was0.29±0.12,0.17±0.06,0.04±0.01,0.26±0.14, respectively. The p53mRNA expression was0.96±0.21,3.84±0.67,1.31±0.23,1.50±0.45, respectively. The p53protein expression was0.041±0.02,0.242±0.10,0.084±0.03,0.057±0.01, respectively.3.2FDG T/M positively correlated with GLUT1. There were no correlation between FDG T/M and p53, VEGF, and HIF-1α.3.3MET T/M positively correlated with CD98, Ki67LI, and MVD before the radiotherapy of tumor.3.4CHO T/M positively correlated with Ki67LI, and MVD before the radiotherapy of tumor.3.5No correlation could be found between ACE T/M and VEGF, HIF-1α. p53, Ki67LI, MVD.Conclusions1. The sensitivity, specificity and accuracy of FDG and MET, CHO PET/CT for detection of brain gliomas recurrence were high. The false positive and negative also exit in FDG and CHO PET/CT for differentiation of brain glioma necrosis and recurrence. MET PET/CT was superior to FDG and CHO in differentiation between the recurrent and necrosis. FDG PET/CT combined with MET or CHO can evaluate the metabolism of brain gliomas in multiple aspects. Multiple tracers PET/CT can improve the accuracy of differentiation of brain glioma necrosis and recurrence.2. Rat ectopic C6glioma model could be established easily, the successful rate was above90%. And the tumor grew rapidly and could be above2cm after2weeks. Rat C6glioma model could be used in morphologic and functional imaging study.3.11C-MET,11C-ACE and18F-FDG PET could sensitively evaluate the effect of radiotherapy, but "C-CHO PET had some false positive.18F-FDG PET was superior to11C radiotracer PET in demonstrating the C6gliomas in the same group.11C-ACE PET was superior to11C-CHO.4. Uptake of18F-FDG PET/CT significantly correlated with tumor size, cell density and GLUT-1.5. Uptake of11C-MET PET/CT significantly correlated with tumor CD98, Ki67LI and MVD before the radiotherapy of tumor and no correlation with Ki67LI and MVD.6. Uptake of " C-CHO PET/CT can reflect Ki67LI and MVD before the radiotherapy of tumor; Uptake of " C-CHO PET/CT correlated with tumor VEGF and MVD and could reflect tumor angiogenesis.7."C-ACE PET/CT T/M could not reflect tumor angiogenesis, Ki67LI, HIF-1α; Uptake of ACE PET/CT correlated with tumor cell density. FDG, MET, CHO and ACE PET/CT could not reflect apoptosis of tumor. |
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