| Objective:Hepatocellular carcinoma (HCC) is one of the most malignant carcinomas inChina. Every year, almost a hundred thousand people die of HCC, and only stomachcancer and lung cancer have higher death rates. Surgery is the main treatment forHCC, but for many patients surgery is not practical due to such factors as: Hepatitis B,hepatocirrhosis, age and hapatosis. And, for some patients following surgery, theirhealth was not as good as expected. With the development of intervention therapy andmedical radiology, treatment for HCC is undergoing change, and international andnational experts have begun to pay more and more attention to these changes.Intervention treatment is comprised of two methods: angiography interventionand non-angiography intervention. Angiography intervention includes: selective,hepatic lobe embolization; super selective, hepatic segments embolization;trans-hepatic, intra-arterial and portal venous pharmacotherapy; selectiveembolization; intra-arterial chemotherapy; and percutaneous, intravascular,port-catheter system chemotherapy. Non-angiography intervention mainly refers toablation therapy, which includes hot ablation, chemical ablation and cryotherapy.During intervention treatment, computer tomography (CT) and magneticresonance imaging (MRI) techniques are necessary, particularly, for checking the inactivation rate of the carcinomas and for detecting residue and carcinomas. Thesetechniques are helpful in evaluating treatment results and for providing post-treatmentinstructions and for enhancing cure rate and life quality. Perfusion weighted imaging(PWI) has been applied successively in clinical diagnoses of central nervous systemresearch in recent years. Application of PWI in liver disease is also receiving moreand more attention from international and national experts, but the application of PWIin follow-up checks after HCC surgery is still based on theoretical research. Clinicalresearch began only a few years ago.Our research purpose was as follows: 1. Using the rabbit VX2 model, thisresearch explores the benefits and possible use of MR, following ablation therapy forHCC. 2. This research also determines the series parameters of MR perfusionfollowing HCC intervention therapy. 3. The research investigated changes in MRparameters before and after HCC intervention therapy. And tissue differences in thetumor area before and after treatment were distinguished, which help checking forcarcinoma residue or recurrence. This research also enhances accurate evaluation ofthe local curing rate of HCC intervention therapy and provides theoretical imagingevidence for HCC clinical treatment plans.Materials and Methods1. Materials1.1 Experimental AnimalsSixteen, adult New Zealand, white rabbits (both males and females from theGuangdong Medical Experimental Animal Center), ranging in weight fromtwo-to-three kg, were divided into four experimental groups (of three rabbits pergroup) and four control groups (with one rabbit in each group).1.2 Establishment of rabbit VX2 liver tumor modelVX2 tumor cells were subcutaneously inoculated in the rabbit thighs to grow into tumors. And the tumors were sectioned and reduced into squares of 1-2mm~2,which could be used for seeding.Rabbits in the experimental groups were laid on CT scanning beds andanaesthetised intravenously with 3% soluble pentobarbitone. Paracentetic needleswere used to process VX2 tumor cells in the left and right liver lobes, with CT guideby. Liver punctures were about 1.5 to 2.0 cm deep and the punctures were sealed withsponge cotton. After recovery and when CT scanning showed no bleeding, the rabbitswere returned to their nests.1.3 Rabbit disposal methodFourteen days after their tumor implants, the rabbits in the experimental groupswere checked by MR every week, until the tumor diameters reached 2.0 cm. At thisstage, PWI was performed first, followed by microwave ablation treatment with CTguide. On the 1st, 7th, 14th and 28th days following ablation, rabbits in both theexperimental and control groups were anaesthetized with 3% soluble pentobarbitone,and plain MRI, PWI and contrast MRI were performed. After MRI, all rabbits werekilled immediately by air embolism, and sections were prepared for pathologicalanalysis and compared with corresponding MR images.1.4 Clinical materialsFrom July 2004 to January 2007, 36 HCC patients were selected. Of these, 31were male and 5 were female. Their ages ranged from 28 to 76 years old, and theaverage age was 54.25±12.68 years. All the patients had plain MRI, PWI and contrastMRI performed before their surgery, and they also had the same treatment performedin the week following surgery. After this, the same treatment was performedonce-a-month in the first three months and once every three months for one yearfollowing their surgery. The patients had follow-up medical check ups for periods ofup to one and a half years. The surgery methods included transcatheter arterial embolization (TAE) (8 cases), microwave ablation (6 cases) and combination of TAEand microwave ablation (12 cases).2. MR scanning methodsAll MR equipments were obtained at a 1.5T MR scanner, provided by theSiemens Company (German).2.1 MR scanning methods of rabbitsThe MR image parameters with FLASH TIWI were as follows: TR 210 ms, TE2.6 ms, section thickness 5 mm, gap 0.6 mm, layers, 10-16, FOV 28 cm X 21cm,matrix 512 X 384, NEX 4, image time 40 s. The MR image parameters with TSET2WI were as follows: TR 4000ms, TE 2101ms, section thickness 5.5 mm, gap 0.6mm, layers 10-16, FOV 28 cm X 21 cm, matrix, 512 X 384, NEX 4, image time 40 s.PWI image parameters with turbo FLASH were as follows: TR 210 ms, TE 1.2 ms,section thickness 5.5 mm, gap 0.6 mm, phase 60, layer 4, FOV 28 cm X 21 cm,matrix 256 X 208, NEX 4, image time 40 s. The contrast agent was fast bolus injectedwith high pressure syringe into ear vein. The contrast agent was Gd-DTPA, the dosewas 0.2mmol/kg and the injection speed was 1ml/s. The contrast agent was injectedright after the scanning started; then, 5 ml salt water was injected to ensure maximumeffectiveness of the contrast agent. MR with FLASH T1WI was carried on after PWI,and the image parameters were the same as plain MRI.2.2 MR scanning methods of human patientsMR image parameters with FLASH T1WI were as follows: TR 107 ms, TE 4.8ms, section thickness 8 mm, gap 0.8 mm, layers 20, FOV 36 cm X 27 cm, matrix 512X 384, NEX 4, image time 40 s. MR image parameters with TSE T2WI were asfollows: TR 3600 ms, TE 101 ms, section thickness 8 mm, gap 0.8 mm, layers 20,FOV 36 cm X 27 cm, matrix 512 X384, NEX 4, image time 40 s.PWI image parameters with turbo FLASH were as follows: TR 210 ms, TE 1.2 ms, section thickness 8 mm, gap 4 mm, phase 20, layer 4, FOV 28 cm X 21 cm,matrix 256 X 208, NEX 4, image time 20 s. The contrast agent was fast bolus injectedwith high-pressure syringe into the ulnar vein. The contrast agent was Gd-DTPA, thedose was 0.2mmol/kg and the injection speed was 3 ml/s. 2 ml contrast agent wasinjected first to establish-the peak time of the agent flow to aortic at the level ofceliac trunk, the scan started 10 seconds before the peak time, then, 20 ml salt waterwas injected to ensure full use of the contrast agent. MR with FLASH T1WI wascarried on after PWI, the image parameters were the same as plain MRI.3. MRI measurement methods and standardsThe largest area of focus was selected to analyze the change of maximum slopeof increase (MSI) and time-intension curve of aorta, spleen, parenchyma, tumor andperiablational enhancement. The regions of blood vessels and artifacts wereprecluded and the largest diameter of the lesions were measured, and the ROI was20±5mm~2 and over 50 pixels.4. Fabrication and analysis standard of pathological changes in rabbits HEAfter MR was performed, all rabbits were killed immediately by air embolism,and rabbit liver sections, 4μm thick, were compared with corresponding MRI. Tumorconditions were observed by eyes, as to color and luster, then, the sections weresoaked and fixed with 10% formaldehyde solution, paraffin embedded and serialsections for pathologic HE were compared with corresponding MRI.Analysis components for pathologic HE sections included: pathologicalhistological types, condition of tumor borders and hemorrhage, coagulation necrosisand fiberhyperplastic of tumor.5. Fabrication and analysis standards of pathological changes in patients HEPathological samples of patients were obtained by surgery or puncture, thesections were soaked and fixed with 10% formaldehyde solution, paraffin embedded and serial sections for pathologic HE were compared with corresponding MRI.6. Statistical analysisAll data in this study were expressed as mean±standard deviation. Statisticalanalysis was based on Student t test with SPSS 14.0 software, and a P value of lessthan 0.05 was adopted to indicate a statistically significant difference. The degree ofvisualization that PWI and conventional MRI sequences revealed residuary orrecurrent tumors after interventional therapy were graded, and analyzed bynonparametric test.Results1. Results of MRI in rabbitsMRI was performed successfully on all the rabbits, and the control group rabbitswere observed not to have abnormalities above T1WI, T2WI and PWI.All 12 rabbits in the four experimental groups grew tumors successfully, and atotal of 20 tumors were found on the 28th day. The tumor diameters of 14 liverlesions in the left lobe and 6 lesions in the right lobe ranged from 1.5 cm to 3.0 cm.All of the tumors in the experimental group were either solitary (in 10 rabbits) ordiffused (in 4 rabbits). A tumor of larger diameter than 2.0cm was selected from eachrabbit for microwave ablation treatment, and twelve tumors were treated all together.MRI was performed on the rabbits on the same day following surgery and, then onthe 7th day, 14th day, and 28th day after surgery. Liver lesions numbered twelve,nine, six and three, respectively, and the tumor diameters were from 2.5 to 3.0 cm.MRI of the experimental group after ablation: on the 28th day after plantingtumor, the diameter was up to 2cm, and the tumors were diffused in liver; theintension of tumors was uneven in T1WI, accompanying necrosis with low intensionin T1WI and hyperintension in T2WI. Part of lesions growed out of liver withlymphadenectasis in abdominal cavity and ascites. The tumors showed heterogeneous enhancement after the injection of contrast agents. Early after RF ablation, the ablatedtumor remained slightly hyperintension T1-and T2-weighted MR images and wassurrounded by a 2-3-mm-thick rim of high signal intensity on T2-weighted images inall cases. On contrast-enhanced MR images, ablated tumors were unenhanced butwere surrounded by an enhancing rim corresponding to the previous hyperintense rimon T2-weighted images. 5-7-mm nodular enhancing abnormalities were noted at theperiphery of the ablated tumor. 7 and 14 days after ablation, the rim enhancementalong the ablated tumor became more prominent compared with that immediatelyafter ablation on contrast-enhanced MR images, and the 28th day after ablation,5-10-mm nodular enhancing abnormalities were noted at the periphery of the ablatedtumor in all cases. 10-15-mm nodular enhancing abnormalities were noted in allcases, and air intension appeared in part of tumors.2. Resulting pathological changes in rabbitsNo tumor or pathological changes were observed in the control group.Pathological HE results in the experimental groups were as follows: round oroval necrosis was observed in tumor tissue. The rim enhancement corresponded to themixture composed of inflammatory reaction with hyperemia and granulation tissue;nodular enhancing abnormalities corresponded to residual viable tumor in all casesand fiber hyperplasia occurred between cancer nests.3. Quantitative studies of PWI in rabbitsThe mean MSI of the control group and the experimental groups liver were18.12±0.46 and 18.92±1.05. The mean MSI of the experimental groups tumors beforeand after surgery were 13.60±0.70 and 13.71±0.73. On the same 7th, 14th, and 28thdays following surgery, the mean MSI of the experimental groups tumors were13.71±0.69, 13.52±0.70, 12.33±0.72and 11.03±0.62, respectively. On the same 7th,14th, and 28th days following surgery, the mean MSI of the benign enhancement tissue were 12.27±0.52, 11.27±0.63, 10.96±0.52 and 10.06±0.58, respectively. Thetime-intension curves of the tumors were observed to ascend rapidly in reaching thepeak, whereas, the enhancement tissue curves ascend slowly to reach the peak.The data showed no significant difference in MSI between the control groupliver and the experimental groups liver after surgery (t=2.11, P＞0.05) and betweenthe experimental groups tumors before and after surgery(t=1.97, P＞0.05). There weresignificant differences between MSI of the experimental groups tumors after surgeryand the benign enhancement tissues (t=13.74, P＜0.05).4. Results of MRI in patientsAmong the 36 HCC patients, there were 8 patients with small carcinomas(diameter of tumor less than 3.0 cm) and 28 patients with large carcinomas (diameterof tumor larger than 10 cm). The result of MRI before surgery was hypointension onT1WI and hyperintension on T2WI, necrosis was observed in some of the largecarcinomas HCC, and the intension was lower than parenchyma. All the smallcarcinomas HCC patients had microwave ablation treatment, and round or ovalnecrosis was observed and hyperintension on T1WI and on T2WI, with circleenhancement one week after the surgery. One month after ablation, the intension ofnecrosis was degraded on T1WI and clearer bouncary. All the large carcinomas HCCpatients had microwave ablation combined TAE treatment, and round or oval necrosiswas observed and hypontension on T1WI and hyperintension on T2WI, with circleenhancement after the surgery.5. Resulting pathological changes in patientsThe HE pathological reSults in patients were as follows: the focus of ablationshowed coagulation necrosis after one month and colliquative necrosis after onemonth. The focus in patients who had TACE treatment showed colliquative necrosis,and the enhancement area showed fiber hyperplasia, in which cancer cells were found among seven patients. The enhancement node was tumor residue, fiber hyperplasiaoccurred between cancer nests.6. Quantitative studies of PWI in patientsThe mean MSI of parenchyma and HCC before surge were 4.80±0.56 and7.81±0.43, and the mean MSI between parenchyma after ablation and benignenhancement area were 4.31±0.46 and 3.75±0.26. With the tumor residue, the meanMSI in parenchyma, enhancement area and the residue tumor were 4.74±0.46,3.70±0.36 and 7.76±0.45. The time-intension curve of residue tumor was observed toascend rapidly to reach the peak, whereas that of the enhancement tissue ascendedslowly to reach the peak.The data showed that there was no significant difference in mean MSI betweenparenchyma before and after surgery and HCC (t=4.51, 1.97, P＞0.05). There was asignificant difference in mean MSI between residue tumor after surgery and thebenign enhancement area (t=39.03, P＜0.05).Capability of PWI on early detection of residuary or recurrent tumor aftersurgery and the semi-quantificational analysis: The degree of visualization that wereshowed by PWI and conventional MRI were divided into 0-3 grades, and were7,11,17 and 26 on PWI and 20,14,16 and 11 on conventional MRI. There was significantdifference in the data analyzed by nonparametric mann-whitney test (Z=3.41,P＜0.05)Diagnosing test of PWI on detection of residuary or recurrent tumor afterablation.72 cases were evaluated by PWI after ablation. 61 cases were proved to betumor residua or recurrence in which 54 cases were diagnosed by PWI accurately,and 7 cases were missed.11 cases were proved to be complete ablation in which 3cases were mistakenly diagnosed by PWI. The sensitivity and specificity of PWI ondetection of residuary or recurrent tumor was 0.89 and 0.73 respectively. Conclusion:1. Conclusion from the rabbit experimentsRegular MRI did not help to distinguish the difference between the benignenhancement area after ablation and the residue tumor. There were no significantdifferences in MSI between the control group and the experimental groups aftersurgery and among the experimental groups before and after surgery, which mean thatthis surgery did not affect the stability of MSI in evaluating the parenchyma andtumor before and after surgery. There was significant difference in MSI among theexperimental groups after surgery and the enhancement tissues, which indicates thatMSI is valuable in distinguishing differences between the benign enhancement areaafter ablation and the residue tumor.2. Conclusion from patients' treatmentRegular MRI did not help to distinguish the difference between the enhancementarea after ablation and the residue tumor, whereas PWI did help. There were nosignificant differences in mean MSI between parenchyma before and after surgeryand HCC, which indicate that surgery does not affect the stability of MSI inevaluating the parenchyma and tumor before and after surgery, and that MSI can beused to evaluate the effect of ablation surgery. There was significant difference inmean MSI between residue tumor after surgery and the benign enhancement area,which indicates that MSI is valuable in distinguishing the difference between theenhancement area after ablation and the residue tumor.This research with the rabbit VX2 HCC model shows that PWI is a verysensitive imaging technique that can be used to distinguish liver tissue condition afterablation surgery. This research also proved that PWI contributed to early stagediagnosis and dynamic monitoring following HCC ablation surgery, which regularMRI is unable to do. MSI can also provide quantitative evidence of pathological change. In clinical applications, PWI can help to diagnose and discriminate diagnosedliver disease.
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