Detection of Cholangiocarcinoma in Primary Sclerosing Cholangitis by Positron Emission Tomography

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1 Detection of Cholangiocarcinoma in Primary Sclerosing Cholangitis by Positron Emission Tomography SUSANNE KEIDING, 1,2 SØREN B. HANSEN, 1 HENRIK HØJGAARD RASMUSSEN, 2,4 ANTONY GEE, 1 AKSEL KRUSE, 3 KLAUS ROELSGAARD, 1,3 ULRIK TAGE-JENSEN, 4 AND JENS FREDERIK DAHLERUP 2 Primary sclerosing cholangitis (PSC) predisposes to cholangiocarcinoma (CC), which usually is widespread in the liver at the time of the diagnosis and which has a median survival of approximately 6 months. Positron emission tomography (PET) is a noninvasive scanning method that allows the assessment of metabolism in vivo by means of positron-emitting radiolabeled tracers. [ 18 F]Fluoro-2-deoxy- D-glucose (FDG) is a glucose analogue that accumulates in various malignant tumors because of their high glucose metabolic rates. The purpose of the study was to develop a PET method to detect small CC tumors in patients with PSC. PET scanning of the liver was performed after intravenous injection of 200 MBq FDG in 9 patients with PSC, 6 patients with PSC CC, and 5 controls. The scanning was performed at successive time intervals for a total of 90 minutes with simultaneous successive arterial blood sampling for radioactivity concentration determination. In each of the PSC CC patients, 2 to 7 hot spots were seen, with volumes of 1.0 to 45 ml (median, 4.4 ml). There were no hot spots in the two other patient groups. The localization of hot spots was confirmed by single-blind evaluation. Data were analyzed by the Gjedde-Patlak plot, yielding values of the net metabolic clearance of FDG, K [ml min ml 1 tissue]. In the CC hot spots, maximum K values were 1.59 to 4.17 (median, 2.34; n 6); in the reference liver tissues of these patients, K values were 0.40 to 0.69 (median, 0.49); in PSC patients, they were 0.23 to 0.53 (median, 0.36); and in controls, they were 0.20 to 0.34 (median, 0.31). The difference between K in CC hot spots and the other groups was statistically significant (P F.001). We conclude that FDG-PET seems to be able to detect small CC tumors and may be useful in the therapeutic management of PSC. (HEPATOLOGY 1998;28: ) Primary sclerosing cholangitis (PSC) is a chronic cholestatic liver disease characterized by inflammation and fibrosis Abbreviations: PSC, primary sclerosing cholangitis; CC, cholangiocarcinoma; PET, positron emission tomography; FDG, [ 18 F]fluoro-2-deoxy-D-glucose; ROI, region of interest; VOI, volume of interest; K, net metabolic clearance of FDG (ml plasma min ml 1 tissue). From the 1 PET Center, 2 Department of Medicine V, 3 Department of Surgery L, Aarhus University Hospital, and 4 Department of Medicine M, Aalborg Hospital, Aalborg, Denmark. Received January 30, 1998; accepted May 4, Supported by grants from the Danish Cancer Society ( ). Address reprint requests to: Susanne Keiding, M.D., PET-Center, Aarhus University Hospital, DK-8000 Aarhus C, Denmark. Fax: Copyright 1998 by the American Association for the Study of Liver Diseases /98/ $3.00/0 of the intrahepatic and extrahepatic bile ducts. It predisposes to the development of cholangiocarcinoma (CC). From selected cohorts of PSC patients 1-5 with a total number of 536 patients, the average prevalence of this neoplasm was calculated to be 9%. The time from the PSC diagnosis until recognition of CC ranged from 1 to 25 years. 2,5,6 CC complicating PSC is usually diagnosed at an advanced tumor stage, with an estimated median survival of 5 to 7 months after the CC diagnosis was made. 6,7 Prognostic models, 1,4,7,8 cancer-associated CA19-9, carcinoembryonic antigen, 9,10 as well as the observation of increased risk of CC in patients with extensive ulcerative colitis, 2 have proved useful in studying the statistical risk of development of CC in PSC patients, but are of limited use in predicting the risk in individual patients. Survival after liver transplantation of patients with established CC is very poor, and most transplantation centers do not accept such patients for liver transplantation. Nevertheless, on the basis of data published from a number of transplantation centers, 3,8,11-13 we calculated that biliary carcinoma was present in 11% of a total of 289 transplanted PSC patients. The diagnosis was made incidentally by examination of the explanted liver in 8% of the patients. In the largest study, 12 the median survival after transplantation was 14 months for 7 patients with CC diagnosed before the transplantation and 23 months for 11 patients with CC diagnosed incidentally. Case reports of long-term survival of 6 years 12 and 8 years 3 after transplantation of PSC patients with incidental CC diagnosis have been reported. It is clear that a noninvasive method that could be used to detect small (and early) CC tumors in patients with PSC would be of considerable advantage for both screening and individual pretransplantation evaluation of this group of patients. Positron emission tomography (PET) is a noninvasive scanning method to assess the metabolism in various organs in vivo by means of positron-emitting radiolabeled tracers. [ 18 F]Fluoro-2-deoxy-D-glucose (FDG) is a glucose analogue (radioactive half-life of 109 minutes) that is phosphorylated in the cells but not further metabolized. Tumor cells have high glucose metabolic rates as first noted by Warburg 14 and accumulate phosphorylated FDG, visible as hot spots, as reported, for example, in patients with liver metastases from pancreatic cancer 15 and colon cancer, 16 and in 3 patients with massive infiltration of CC. 17 In contrast to other cells, including bile duct cells, hepatocytes possess a high glucose- 6-phosphatase activity. We therefore expected a rapid turnover of tracer in the hepatocytes in contrast to accumulation in CC cells. FDG-PET therefore should be especially suited 700

2 HEPATOLOGY Vol. 28, No. 3, 1998 KEIDING ET AL. 701 for the detection of CC tissue as hot spots in the liver after administration of FDG. The purpose of the present study was to develop a PET method for detection of small CC tumors in patients with PSC. PATIENTS AND METHODS Patients. Nine patients with PSC and with no CC (called PSC patients), 6 patients with PSC and CC (called CC patients), and 5 patients with no liver disease (called control patients) participated in the study. The PSC diagnosis was based on typical cholangiographic findings of multiple strictures and dilatations of the infra- and/or extrahepatic bile ducts shown by endoscopic retrograde cholangiography and liver histology consistent with PSC, in accordance with published diagnostic criteria. 18,19 PSC was associated with ulcerative colitis in 8 individuals and Crohn s disease in 4 individuals. The suspicion of CC was raised because of icterus and ultrasound examination in 3 patients, and by endoscopic retrograde cholangiography in 3 patients. The CC diagnosis was based on histological examination of liver biopsies. None of the liver patients had signs of gallstones, pancreatic diseases, or previous operations in the bile ducts. None of the patients had cirrhosis, esophageal varices, ascites, encephalopathy, or signs of cancer except the CC. None of the patients classified as having PSC without CC were found to have clinical or biochemical signs of malignancies during a follow-up period of 13 to 31 months. The ages of the patients in the groups of CC patients and PSC patients were 46 to 74 years (median, 62 years) and 27 to 51 years (median, 36 years), respectively. The respective male/female ratio was 4/2 in CC and 7/2 in PSC. The CC patients and PSC patients had plasma albumin of 374 to 576 µmol/l (median, 484 µmol/l) and 422 to 667 µmol/l (median, 606 µmol/l), respectively. The CC patients and PSC patients had plasma bilirubin of 20 to 364 µmol/l (median, 92 µmol/l) and 7 to 71 µmol/l (median, 20 µmol/l), respectively. Plasma coagulation factors were normal in all patients. At the time of the PET scanning, none of the patients had fever or received antibiotics. The control patients were women with a breast tumor in whom a PET scan with FDG was performed for diagnostic purposes. The PET protocol was identical to that used in the liver patients (see below), with the exception that these patients were located with the breast within the 15-cm field of view of the scanner (see below). Part of the liver in these patients was within the scanner field of view. This part was large enough to allow for definition of liver regions of interest by a procedure identical to that used in the liver patients. The ages of the control patients were 61 to 83 years (median, 65 years), and they had no signs of cancer except for in the breast and, in some individuals, in the axillary lymph nodes. Liver biochemistry as mentioned above was normal. Before the PET examination, the patients fasted overnight but were allowed to drink water. Blood glucose was 5.1 to 8.3 mmol/l (median, 5.6 mmol/l), 5.0 to 5.3 mmol/l (median, 5.1 mmol/l), and 4.0 to 5.8 mmol/l (median, 4.5 mmol/l) in CC, PSC, and controls, respectively. This did not change significantly or systematically during the examinations. Ethics. The study was approved by the Ethics Committees for the Hospitals in Aarhus and North Jutland Counties. The radioactivity dose received by the patients was, on the average, 5.5 msv, corresponding to twice the background radioactivity per year in Denmark. Informed consent to the procedures was obtained from each subject. No complications were observed. PET Examination. The patients were positioned in the 60-cm opening of the Siemens ECAT EXACT HR (961) scanner so that the xiphoid process was located in the center of a 15-cm field of view (and with the bed position as low as possible). For the control patients, see above. The scanning was performed with 47 axial slices each 3.1 mm in the 15-cm field of view, yielding radioactivity concentration measurements in volumes of mm 3 (voxel). FDG was given as an intravenous injection of 197 to 283 MBq (median, 214 MBq; corresponding to less than 40 pmol; radiochemical purity 98%) into a brachial vein in 4 to 5 ml isotonic saline, and was flushed immediately after with 20 ml saline. The scanning started with a 15-minute transmission scan acquired before the injection of FDG and used for attenuation correction. After the FDG injection, a dynamic PET scan of the liver was performed for a total of 90 minutes with the following frame sequences: 10-second scans for 3 minutes, 60-second scans for 7 minutes, 2-minute scans for 10 minutes, 5-minute scans for 20 minutes, and 10-minute scans for 50 minutes. The data were reconstructed using a Hann filter with a cutoff frequency of 0.3, resulting in a uniform resolution with a full width at half maximum (FWHM) of 6.7 mm. The data were recorded as integrated mean values in each of the time frames and corrected for radioactivity decay to the start of the tracer injection. Blood samples of each 1 ml were taken for radioactivity concentration measurements from a percutaneously placed catheter in a radial artery over a period of 90 minutes, as follows: every 10th second for 3 minutes, 30th second for 2 minutes, 60th second for 5 minutes, 5th minute for 35 minutes, and 15th minute for 45 minutes. The blood samples were taken in the course of approximately 4 seconds (and not just 1 second) to approximate integrated concentration measurements. The volume of the sampling catheter was 0.10 ml, and immediately before sampling, approximately 0.90 ml blood was drawn to flush the catheter. Blood was centrifuged, and the plasma radioactivity concentration was measured in a well counter and corrected for radioactivity decay to the start of the tracer injection. Image Analysis. Visual analysis of the scanning data was performed in images of transaxial slices consisting of the mean radioactivity concentration in frames from 12 to 90 minutes after the injection. Within the 15-cm field of view, the whole liver was seen, with the heart visible in the upper slices and the kidneys in the lower. In some of the patients, the spleen could also be localized. The position of the intrahepatic part of the caval vein was localized in the first time frame (where the radioactivity appeared rapidly following the intravenous tracer injection). In each transaxial slice of these data, the borders of the liver were defined conservatively with respect to these extrahepatic structures. The transaxial slices were subsequently examined visually to define hot spots (radioactivity accumulation). Figure 1 gives an example. For each hot spot, the voxel containing the highest radioactivity concentration was identified. Regions of interest (ROI) were drawn using an isocontour that enclosed all voxels having at least 75% of the maximum radioactivity concentration. In adjacent slices, ROI were similarly defined using the same maximum voxel. Adjacent ROI were summed to form a volume of interest (VOI). The VOI of the hot spots (CC-VOI) were copied to the transaxial slices of the dynamic studies, characterized by a specific volume (ml tissue) and an average radioactivity concentration (kbq/ml tissue) for each time frame. For each CC patient, two CC-reference VOI with no apparent radioactivity accumulation were defined, as judged visually by one of the investigators (S.K.). For each PSC patient, two PSC-VOI, and for each control patient, two control-voi were defined. It was checked that the radioactivity concentration measurements did not depend systematically on the size of the VOI or on the localization within different slices. The localization of hot spots in the livers (by author S.K.) were checked by the single-blind evaluation of another author (K.R.). For each VOI, a time-activity curve of the tissue radioactivity concentration was estimated. Figure 2 gives examples. The timeactivity curves of the VOI were analyzed in relation to the respective plasma time-activity curves by the Gjedde-Patlak plot analysis (see legend of Fig. 3). By this analysis, an estimate of the net metabolic clearance of FDG, K, (ml plasma min 1 ml 1 tissue) was obtained for each VOI. For each patient, the mean of the two K values in CC-reference VOI, PSC-VOI, and control-voi were used in the further analyses.

3 702 KEIDING ET AL. HEPATOLOGY September 1998 FIG. 1. PET image of the liver with a hot spot. Transaxial slice showing the mean tissue radioactivity concentration 12 to 90 minutes after intravenous injection of 225 MBq FDG to a patient with PSC and CC (case P.E.L.). The image demonstrates a hot spot (red) in surrounding liver tissue (blue). FIG. 2. Time-activity curves. Time courses of radioactivity concentrations in a CC-VOI ( ), CC-reference VOI ( ), and plasma ( ) (case P.E.L. with PSC CC). The radioactivity concentration is corrected for radioactivity decay back to the time of the start of the tracer injection.

4 HEPATOLOGY Vol. 28, No. 3, 1998 KEIDING ET AL. 703 FIG. 3. Gjedde-Patlak plots of the radioactivity concentrations in a CC-VOI ( ) and a CC-reference VOI ( ) (case P.E.L. with PSC CC) using data from the time-activity curves in Fig. 2. The curves show the relation between the tissue radioactivity concentration at time t, M(t), [kbq/ml tissue], normalized with respect to the plasma radioactivity concentration at time t, c p (t), [kbq/ml plasma], versus the virtual time, t cp d M(t) c p (t) 0 c p (t) The intercept of the y-axis is the volume of distribution of FDG, V g [ml plasma/ml tissue]. The net metabolic clearance of FDG, K [ml plasma min 1 ml 1 tissue] was calculated as the slope of the linear regression of M/c p on the virtual time for the data points from 12 to 90 minutes after the injection, where there was a linear relationship, indicating a constant rate of accumulation of phosphorylated FDG in the tissue. 21 K V g Statistics. Analysis for measurement uncertainty (repeatability) was performed by comparison of the two estimates of the K values within each patient by means of the within measurement coefficient of variation. 23 Analyses for statistically significant differences of the K values between the groups were performed by the Kruskal-Wallis and the Mann-Whitney tests. RESULTS In each of the CC patients, between 2 and 7 hot spots were defined in the liver (Fig. 1). The volume of the CC-VOI ranged from 1.0 ml to 45 ml (median, 4.4 ml). The VOI were of irregular shape, but if looked upon as spheres, the diameters were 1.0 cm to 3.6 cm (median, 2 cm). There were no hot spots detectable in any of the PSC or control patients. There was complete agreement between the open analysis with knowledge of the patient diagnoses (S.K.) and the single-blind analysis (K.R.). Figure 2 gives an example of the time-activity curves. The plasma radioactivity concentration curve showed an initial peak, followed by a smaller peak of the reference tissue curve. The two curves crossed after about 30 minutes, so that the reference tissue curve became higher than the plasma curve after this time. The CC-VOI time-activity curve showed a similar initial peak but then did not decrease in the course of the measurement period. Figure 3 shows the Gjedde-Patlak plot, using the data depicted in Fig. 2. It is seen that the last parts of the curves were approximately linear. The slope of the linear part of the curve (last 13 points) was higher in the CC-VOI than in the

5 704 KEIDING ET AL. HEPATOLOGY September 1998 CC-reference VOI in each of the CC patients. The net metabolic clearance of FDG, K, was calculated according to the equation in the legend of Fig. 3. The measurement uncertainty as calculated from the two K values in each patient was 21% for the control group, 24% for the PSC group, and 21% in the CC-reference group. In the further analyses, the individual mean values were used. Figure 4 shows the values of the net metabolic clearance of FDG, K. In the CC-VOI, the highest individual K values were 1.59 to 4.17 (median, 2.34; n 6). In the CC-reference VOI, the individual mean K values were 0.40 to 0.69 (median, 0.49; n 6). In the PSC patients, K was 0.23 to 0.53 (median, 0.36; n 9). In the control patients, K was 0.20 to 0.34 (median, 0.31; n 5). The Kruskal-Wallis and the Mann- Whitney tests showed that the K values from the control group were statistically significant from each of the three other groups (P.01), and that the K values from the CC-VOI were significantly different from each of the three other groups (P.001); there was no statistically significant difference between the K values from the control-voi and the PSC-VOI (P.05). The ratio between the highest individual K values from the CC-VOI and the CC-reference VOI were 3.0 to 9.1 (median, 4.9). DISCUSSION The values of the net metabolic clearance of FDG (K) distinguished clearly between malignant CC tissue and nonmalignant tissue. There was no overlap of the K values, and there was even an interval between the lowest CC value and the highest values in the PSC and control tissues (see Fig. 4). There were no false-positive or false-negative values. The smallest value of the estimated tumor volume was 1.0 ml (diameter, 1.0 cm), so the method may be reasonably sensitive for clinical diagnostic use. On the other hand, there was a tendency for smaller K values in VOI with the smallest tumor volumes, probably because of the limitation of the resolution of PET (partial volume effect). The present definition of the hot spots may furthermore be biased by the fact that they were found in livers in which there were larger hot spots. The potential of PET to detect small tumors depends on a high metabolic activity. Thus, a tumor that is smaller FIG. 4. The net metabolic clearance of FDG, K [ml plasma min 1 ml 1 tissue] in 5 control patients (Control) ( ), 9 PSC patients (PSC) ( ), and 6 CC-reference VOI (CC-ref) ( ). In the 6 CC patients, K values from the same patient are connected by straight lines, and the key-k values (maximum values) are shown with filled symbols ( ), the others with open symbols ( ).

6 HEPATOLOGY Vol. 28, No. 3, 1998 KEIDING ET AL. 705 than the PET resolution of 5 to 7 mm but with a very high metabolic activity may be diagnosed, whereas a larger tumor with less metabolic activity may be missed. The significantly higher K values in CC-reference tissue and in the PSC patients without CC compared with the controls may suggest that there is an increased metabolic rate in the PSC tissue. This is of potential importance for the investigations of a possible multiple origin of the CC in patients with PSC. 13,19 Okazumi et al. 17 examined 3 patients with large CC ( 3 cm) by PET and calculated rate constants for the enzymatic reactions according to a four-compartment model. Other authors have applied various three-compartment models 24 to PET studies of FDG kinetics in normal human livers. We analyzed the time-activity curves by means of the Gjedde- Patlak plot analysis (Fig. 3). This calculation procedure only assumes irreversible metabolic trapping of radiolabeled metabolites and is based on observable quantities (Fig. 2), with fewer assumptions concerning input functions and distribution of the tracer than the aforementioned three- and four-compartment models. The Gjedde-Patlak analysis is used to quantify the FDG metabolism in the brain, 25 but to our knowledge, it has not previously been applied to the liver. A fuller evaluation of the liver PET-FDG model will be made in a subsequent article (Keiding S, unpublished data, January, 1998). It may be noted that there were no significant differences between the blood glucose concentrations between the three study groups (see Patients and Methods). This is important in view of the higher K values found in the liver after glucose intake than in the fasting state calculated from a threecompartment model. 24 As mentioned in the introduction, PET-FDG is probably especially well suited to detect CC in the liver, because not only does radioactivity accumulate in the malignant bile duct cells, but there is also a rapid turnover of the radioactivity in the surrounding hepatocytes (see Fig. 2). For tumors with sufficiently increased metabolism, PET therefore may be better suited to the detection of small and early malignant tumors than morphological imaging techniques, such as ultrasound, computed tomography, and magnetic resonance scanning. In the present study, the conventional imaging analyses were not performed with emphasis of reporting the size and exact number of tumor-suspected changes, and therefore a direct comparison cannot be performed. Nevertheless, retrospective reading of the patient files revealed that in 3 of the 6 CC patients, PET hot spots were found in liver regions where ultrasound examination (and in 1 patient, magnetic resonance) had not detected morphological changes. Increased incidence of hepatocellular carcinoma has also been reported in PSC patients 2. Preliminary and conflicting results concerning the efficiency of PET to detect this type of tumor have been published, 17,26,27 but it seems as if PET-FDG can detect low-differentiated, but not well-differentiated, tumors, probably because of similarities between the metabolism of FDG in normal hepatocytes and tumor cells deriving from hepatocytes. From a clinical point of view, the introduction of a new test is, strictly speaking, only justified if the use of it leads to significant changes in the therapeutic management of the individual patient. 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