Transarterial chemoembolization (TACE) is the

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1 Efficacy of Selective Transarterial Chemoembolization in Inducing Tumor Necrosis in Small (<5 cm) Hepatocellular Carcinomas Rita Golfieri, 1 Alberta Cappelli, 1 Alessandro Cucchetti, 2 Fabio Piscaglia, 3 Maria Carpenzano, 1 Eugenia Peri, 2 Matteo Ravaioli, 2 Antonia D Errico-Grigioni, 4 Antonio Daniele Pinna, 2 and Luigi Bolondi 3 Transarterial chemoembolization (TACE) is commonly used as a bridge therapy for patients awaiting liver transplantation (LT) and for downstaging patients initially not meeting the Milan criteria. The primary aim of this study was to analyze whether a difference exists between selective/superselective and lobar TACE in determining tumor necrosis by a pathological analysis of the whole lesion at the time of LT. The secondary aim was to investigate the relationship between the tumor size and the capacity of TACE to induce necrosis. Data were extracted from a prospective database of 67 consecutive patients who underwent LT for hepatocellular carcinoma and cirrhosis from 2003 to 2009 and were treated exclusively with TACE as a bridging (n 5 53) or downstaging therapy (n 5 14). We identified 122 nodules; 53.3% were treated with selective/superselective TACE. The mean histological necrosis level was 64.7%; complete tumor necrosis was obtained in 42.6% of the nodules. In comparison with lobar TACE, selective/superselective TACE led to significantly higher mean levels of necrosis (75.1% versus 52.8%, P ) and a higher rate of complete necrosis (53.8% versus 29.8%, P ). A significant direct relationship was observed between the tumor diameter and the mean tumor necrosis level (59.6% for lesions < 2 cm, 68.4% for lesions of cm, and 76.2% for lesions > 3 cm). Histological necrosis was maximal for tumors > 3 cm: 91.8% after selective/superselective TACE and 66.5% after lobar procedures. Independent predictors of complete tumor necrosis were selective/superselective TACE (P ) and the treatment of single nodules (P ). Repeat sessions were more frequently needed for nodules treated with lobar TACE (31.6% versus 59.3%, P ). Conclusion: Selective/superselective TACE was more successful than lobar procedures in achieving complete histological necrosis, and TACE was more effective in 3- to 5-cm tumors than in smaller ones. (HEPATOLOGY 2011; 53: ) Abbreviations: CEUS, contrast-enhanced ultrasonography; CT, computed tomography; HCC, hepatocellular carcinoma; LT, liver transplantation; MC, Milan criteria; MRI, magnetic resonance imaging; PEI, percutaneous ethanol injection; TACE, transarterial chemoembolization. From the 1 Radiology Unit, 2 Liver and Multiorgan Transplant Unit, 3 Division of Internal Medicine, Department of Digestive Diseases and Internal Medicine, and 4 Pathology Division, Felice Addarii Institute, Department of Oncology and Hematology, Sant Orsola-Malpighi Hospital, University of Bologna, Bologna, Italy. Received October 1, 2010; accepted February 3, Address reprint requests to: Alberta Cappelli, M.D., Unità Operativa Radiologia Malpighi, Azienda Ospedaliero-Universitaria di Bologna, Policlinico Sant Orsola-Malpighi, Via Albertoni 15, Bologna, Italy. alberta.cappelli@aosp.bo.it; fax: þ Copyright VC 2011 by the American Association for the Study of Liver Diseases. View this article online at wileyonlinelibrary.com. DOI /hep Potential conflict of interest: Nothing to report. Additional Supporting Information may be found in the online version of this article Transarterial chemoembolization (TACE) is the recommended treatment strategy for patients with intermediate-stage hepatocellular carcinoma (HCC) according to the American Association for the Study of Liver Diseases guidelines. 1,2 In the setting of liver transplantation (LT), TACE is applied both to reduce the dropout rate for patients on the waiting list (bridge therapy) and to downstage patients with HCC not initially meeting the transplantability criteria (downstaging protocols). 3 The capability of TACE to induce extensive tumor necrosis is still under debate, and this technique is considered to be a noncurative modality. Whether this belief derives from the real potential of the technique or from the fact that it has mainly been applied to tumors that are large and are, consequently, more difficult to treat is still a matter of discussion. Similarly, the role of the various technical modalities of TACE procedures in determining the final rate of necrosis has not been adequately investigated in Western countries. The recommendation for TACE as the standard of care for intermediate-stage HCC is based on the

2 HEPATOLOGY, Vol. 53, No. 5, 2011 GOLFIERI ET AL demonstration of improved survival in comparison with the best supportive care or suboptimal therapies in a meta-analysis of six randomized control trials. 4 However, there was considerable heterogeneity between the individual study designs of the six trials, and the differences included the patient populations and TACE techniques. More specifically, the oldest trials of the meta-analysis included lobar or whole liver embolization (i.e., the injection of a mixture of Lipiodol, a chemotherapeutic agent, and an embolizing material into either the main lobar artery or the hepatic artery itself), whereas more recently, selective treatments have been used (i.e., the injection of agents into the segmental or subsegmental branches feeding the tumors) with apparently better survival results. 5,6 On the basis of radiological features, selective/superselective TACE has already been shown to be capable of obtaining a higher rate of tumor necrosis than conventional lobar or whole liver TACE. 7 However, whether the findings of improved survival with selective techniques really correspond to an improved necrotizing capability, reduced liver toxicity, or both has never been elucidated on the basis of histological findings in a sufficiently large Western population. The results of studies published in the Asiatic literature suggest that segmental or subsegmental TACE has been more effective and has resulted in higher rates of tumor necrosis (64%-83%) than proximal/whole liver TACE (approximately 38%) in historical series Even though the efficacy of TACE can be reliably assessed only by the measurement of tumor necrosis during a histological examination of the whole tumor, only three of these series 8,10,11 included surgically removed nodules, and the histological quantification of necrosis involved small sample sizes (11, 12, and 7 lesions, respectively). However, in the Western literature, the advantages of selective embolization have not been well reported because nonselective TACE has been performed even in recent studies. 12 Therefore, the primary aim of this study was to analyze whether a difference exists between selective/superselective and lobar TACE in determining tumor necrosis by a pathological analysis of the whole lesion at the time of transplantation. The secondary aim was to investigate the relationship between the tumor size and the capacity of TACE to induce necrosis. Patients and Methods Data were extracted from a prospectively collected database for 118 consecutive patients who had a pretransplant diagnosis of HCC resulting from cirrhosis, underwent LT between January 1, 2003 and December 31, 2009 at the Liver and Multiorgan Transplant Unit of Sant Orsola-Malpighi Hospital, and were treated with bridging or downstaging procedures. The final study population consisted of 67 patients treated only with TACE (performed exclusively at our tertiary care institution), as outlined in Fig. 1 and Table 1, with 53 patients meeting the Milan criteria (MC) and 14 meeting our downstaging protocol. 3,13 Before undergoing TACE, all patients were assessed (1) to define the degree of liver function by laboratory examinations and (2) to detect and characterize all liver nodules by imaging techniques. The Child-Pugh score and the Model for End- Stage Liver Disease score (the latter according to the formula proposed by Freeman et al. 14 ) were calculated. The patients were staged according to the United Network for Organ Sharing guidelines 15 and the integrated Barcelona Clinic Liver Cancer staging system. 16 Our downstaging protocol is based on radiological findings at the initial assessment of (1) a single HCC 8 cm, (2) bifocal HCCs < 5 cm, or (3) up to five nodules (each 4 cm) with a total tumor diameter 12 cm, as previously reported. 4,13 The last patients were listed for LT once their HCCs were successfully downstaged to meet the MC. The criteria for successful downstaging were based at that time only on the maximum diameter of tumors with imaging signs of vital tissue, whatever its extent within the tumors was. 1,2,17 Exclusion criteria from the waiting list included evidence of gross vascular invasion, tumor progression beyond the limits of the MC, and evidence of extrahepatic or lymph node metastases. Portal thrombosis was not an exclusion criterion if it could be shown to be nonneoplastic. 18 Imaging Evaluation. Since 2003 (when the study began), our technical requirements for contrastenhanced computed tomography (CT) and magnetic resonance imaging (MRI) have met the minimal criteria subsequently recommended by the American consensus on the diagnostic assessment of liver nodules in patients on the waiting list for LT. 19 For CT, four contrast phases were carried out after precontrast scans (early and late arterial, venous, and late), whereas only three phases were carried out for MRI (arterial, venous, and late). The diagnosis was established according to the latest international guidelines on the management of HCC (i.e., the European Association for the Study of the Liver guidelines from and the American Association for the Study of Liver Diseases guidelines from ,2 ). Whenever needed, CT or MRI was used along with low mechanical index

3 1582 GOLFIERI ET AL. HEPATOLOGY, May 2011 total volume of 50 ml (range ¼ ml) was injected under fluoroscopic control, and this was followed by embolization with Spongel (GelitaSpon gel) particles until there was complete stasis in the tumorfeeding vessels. In all cases, there was testing for a parasitic tumor blood supply through accessory arteries (i.e., the inferior phrenic, internal mammary, or intercostal arteries; Fig. 3), and if one was present, the patient underwent additional superselective treatment (a chemotherapeutic mixture plus embolization). Nonselective lobar TACE consisted of the injection of the same treatment material used in the selective procedures into the right or left lobar branches and then embolization (Fig. 4). Consequently, a larger region (usually the whole lobe containing the tumors) was treated. A selective or, if possible, superselective TACE procedure was the preferred modality whenever it was technically feasible. In all other cases (i.e., when there Fig. 1. Flow chart of the study population. Abbreviation: PEI, percutaneous ethanol injection. contrast-enhanced ultrasonography (CEUS) with Sonovue (Bracco, Milan, Italy). Since 2006, all studies have been evaluated with the support of the institutional picture archiving and communication system (Carestream, version 1.1, Kodak, Rochester, NY), and the radiological reports stored in the radiology information system (e-ris, Exprivia Project SpA, Rome, Italy) were used for this study. Before then, the images had instead been printed on the films used by radiologist to make their reports. TACE Protocol. Two different techniques were applied to treat HCC nodules: lobar and selective/ superselective. With the selective/superselective technique, the tumor-feeding arteries were catheterized with a highly flexible coaxial microcatheter (a 2.7- to 2.8-Fr Terumo Progreat microcatheter or a Boston Scientific Renegade HI-FLO microcatheter) passed through a 4-Fr catheter previously placed approximately in the hepatic artery itself. More specifically, for selective TACE, the tip of the microcatheter was placed into the hepatic arterial branch afferent to the segment in which the tumor was located. In the case of superselective TACE, the tip of the catheter was further advanced into the subsegmental branches feeding the tumor (Fig. 2A,B). After microcatheter placement, a mixture of epirubicin (Pfizer) and Lipiodol (Guerbet) with an average Table 1. Baseline Clinical and Tumor Characteristics of the Study Population Variable Value Age at start (years) /57 (37-67) Male gender 60 (89.6%) Serum creatinine (mg/dl) /0.90 ( ) Serum bilirubin (mg/dl) /1.85 ( ) International normalized ratio /1.35 ( ) Child-Pugh class A 54 (80.6%) Model for End-Stage Liver Disease score /12.5 (7-26) Hepatitis B positive serology 16 (23.9%) Hepatitis C positive serology 46 (68.7%) United Network for Organ Sharing T stage at start T1 10 (14.9%) T2 43 (64.2%) T3-T4a 14 (20.9%) Barcelona Clinic Liver Cancer stage at start A (within MC) 53 (79.1%) B (outside MC) 14 (20.9%) Single nodule 32 (47.8%) Number of nodules 2 (1-4) Diameter of largest tumor (cm) /2.7 ( ) Sum of tumor diameters (cm) /3.6 ( ) Serum alpha-fetoprotein (ng/ml) 14 (2-735) Selective/superselective TACE 38 (56.7%) Lobar TACE 27 (40.3%) Combination of selective and lobar TACE 2 (3.0%) Repeat TACE 29 (43.3%) There were 67 patients in this study. Continuous variables are reported as means and standard deviations, medians and ranges, or both; other variables are reported as numbers and percentages. The MC included United Network for Organ Sharing stages T1 and T2. The tumors were classified as T3 to T4a and fulfilled previously published downstaging eligibility criteria. 3,13 The only Barcelona Clinic Liver Cancer stages were A and B (which corresponded to those fulfilling the MC and those not fulfilling the MC, the latter being intermediate HCC patients meeting our downstaging criteria) because no patient with portal or hepatic vein infiltration or with distant metastasis is eligible for transplantation at our center.

4 HEPATOLOGY, Vol. 53, No. 5, 2011 GOLFIERI ET AL Fig. 2. Hepatic angiographic examination. Left: The cranial portion of this large (>3-cm) HCC of the right hepatic lobe was previously treated (the quite visible Lipiodol accumulation is indicated by the dashed arrow). One month after the first TACE procedure, a residual viable tumor was evident in the peripheral portion of the nodule on a CT scan, and additional TACE treatment was required. A superselective contrast injection into the distal subsegmental branches of the right hepatic artery (the tip of the microcatheter is indicated by the black arrow) showed two small vessels feeding the viable caudal portion (indicated by the empty arrow), which were then subjected to TACE. Right: A postprocedural control showed the complete devascularization of the target nodule with persistent flow into the proximal trunk of the catheterized vessel. was multinodular disease in one lobe with a nodule or nodules fed by multiple arteries or when the catheterization of the tiny tumor-feeding vessels was not possible), lobar TACE was performed. A CT scan was performed approximately 30 days after the procedure to detect both Lipiodol retention within the tumor and residual viable tumor tissue. Homogeneous and dense Lipiodol uptake with no additional contrast enhancement was considered to correspond to a complete response. When this was not the case and residual viable tumors were confirmed by complementary imaging studies (MRI or CEUS) or new lesions had developed but the patients maintained adequate hepatic function and reserve, they were referred for repeat procedures. TACE treatment was repeated on demand, that is, in patients with residual or recurrent tumors observed by CT or MRI, according to the amended Response Evaluation Criteria in Solid Tumors guidelines and in agreement with recent expert opinion. 20 The CT or MRI protocol after a TACE procedure was the same as that applied before TACE. A viable tumor was defined by contrast agent uptake in the arterial phase and washout in the portal phase and/or late phase. During the CT scan, contrast enhancement was visually assessed by a comparison of the unenhanced and arterial phase images to distinguish between iodized oil and contrast agent enhancement. In doubtful cases, CEUS, MRI, or both were performed, as previously described. Histopathology. After LT, all the livers were examined by two experienced hepatobiliary pathologists. The livers were sectioned into 10-mm-thick sections. All identified nodules were grossly described with respect to the site, size, types of margins (vaguely/distinctly nodular or infiltrative), and necrosis, and they were completely paraffin-embedded. Multiple 3-lm sections were stained with hematoxylin and eosin, reticulin, periodic acid Schiff with and without diastase, and Perls iron stain. The percentage of tumor necrosis was calculated as the proportion of the necrotic tissue with respect to the total tumor area and was based on the expert judgment of the pathologist in 10% steps. A necrosis rate of 100% was assumed to indicate complete necrosis; a rate of 99% or less was considered to indicate incomplete necrosis. For the purpose of Fig. 3. Angiographic examination. Selective TACE was performed after catheterization of the proximal trunk of the right inferior phrenic artery (the microcatheter is indicated by the black arrow); this contributed to the perfusion of the HCC (the tumor stain is indicated by the empty arrow) located in the dome of the liver (segment VII; the dome is indicated by the dashed arrow).

5 1584 GOLFIERI ET AL. HEPATOLOGY, May 2011 Fig. 4. Angiographic examination before (left) and immediately after lobar TACE (right). Left: The treatment mixture was injected into the right hepatic artery (the distal part of the catheter in the right hepatic artery is indicated by the black arrow), which also fed the HCC in segment VI (the tumor stain is indicated by the empty arrow). Right: A postprocedural control demonstrated the complete uptake of the Lipiodol-drug emulsion and the disappearance of any perfusion within the tumor along with the absence of perfusion in the peripheral arterial branches of almost the whole right lobe. evaluating the percentage of complete necrosis according to tumor size, the HCCs were grouped by size: 2, 2.1 to 3.0, and 3.1 to 5.0 cm. Statistical Analysis. Continuous variables were reported as means and standard deviations, medians and ranges, or both; the differences between the subgroups were analyzed with the t test after the Levene correction, analysis of variance, or the Mann-Whitney test as appropriate. Categorical variables were reported as numbers and percentages, and the differences between the subgroups were analyzed with the chi-square test with a Yates correction. The amounts of tumor necrosis were reported both as continuous variables and as semiquantitative values, and the differences between subgroups were calculated. In order to identify the potential relationships between the covariates with respect to tumor necrosis, all variables significantly affecting tumor necrosis in the univariate analysis were entered into a multivariate logistic regression model to identify the independent predictors of complete tumor necrosis. A P value < 0.05 was considered statistically significant in all cases. Statistical analysis was carried out with SPSS 13.0 (SPSS, Inc., Chicago, IL). Results The baseline clinical and tumor characteristics of the study group are reported in Table 1. Thirty-eight of the 67 patients underwent selective/superselective TACE exclusively (56.7%), 27 patients underwent lobar TACE exclusively (40.3%), and 2 patients were treated with a combination of the two techniques (3%). In the latter two cases, lobar TACE and selective TACE were each used in only one lobe, and this allowed an assessment of the treatment outcome for each technique. In order to limit the risk of liver decompensation, we never performed whole lobe treatments of both lobes in the same session (or whole liver treatments). Thirty-eight patients had a single course of TACE (56.7%), and the remaining 29 had two or more courses (43.3%). The median time from the first TACE procedure to LT was 8.7 months (range ¼ 1-32 months), and the median time on the waiting list was 6.2 months (range ¼ 1-29 months). For patients who underwent more than one TACE session, the median time from the last imaging procedure to LT was 2.6 months (range ¼ 1-92 days). No patient of the present series experienced major complications related to the procedure, and none underwent LT within 30 days of the procedure (this could be interpreted as an expression of rapid deterioration of liver function). The median hospital stays were 4.5 days after lobar procedures (range ¼ 2-65 days) and 3.5 days after selective/superselective TACE (range ¼ 2-56 days, P ¼ 0.651); clinically significant fever (maximum temperature > 38 C) occurred in 20 cases after lobar TACE (74.0%) and in 23 cases after the selective procedure (60.5%, P ¼ 0.255). Details regarding the course of liver laboratory tests after TACE are reported in Table 2. Clinical and tumor characteristics with respect to lobar and selective/superselective TACE are reported in Table 3: as expected, the choice of the procedure was affected by the presence of multinodular tumors, but it was unaffected by liver function status, although a minimal trend toward worse liver function in those treated with selective/superselective TACE emerged. Patients who underwent selective/superselective TACE required fewer repeat procedures than patients who underwent lobar TACE [12 of 38 (31.6%) versus 16 of 27 (59.3%), P ¼ ] because residual vital tumors were less common. One of the two patients who received the combination of techniques required one additional treatment. Histological Necrosis According to the TACE Modality. Because the type of TACE performed in each patient was affected by the number of tumors

6 HEPATOLOGY, Vol. 53, No. 5, 2011 GOLFIERI ET AL Table 2. Clinical Course of Liver Function Parameters After TACE With Respect to the Adopted Procedure Variable Baseline Value (n 5 67) Day 1 (n 5 67) Day 3 (n 5 60) Day 5 (n 5 39) Serum creatinine (mg/dl) 0.90 ( ) 0.96 ( ) 0.94 ( ) 0.93 ( ) Serum bilirubin (mg/dl) 1.85 ( ) 2.44 ( ) 2.95 ( ) 2.66 ( ) International normalized ratio 1.35 ( ) 1.39 ( ) 1.40 ( ) 1.40 ( ) Model for End-Stage Liver Disease score 12.5 (7-26) 14 (9-26) 14 (9-27) 14 (9-27) Lobar TACE (n) Serum creatinine (mg/dl) 0.94 ( ) 0.96 ( ) 0.96 ( ) 0.97 ( ) Serum bilirubin (mg/dl) 1.59 ( ) 2.12 ( ) 2.84 ( ) 2.64 ( ) International normalized ratio 1.29 ( ) 1.33 ( ) 1.34 ( ) 1.39 ( ) Model for End-Stage Liver Disease score 12 (8-19) 13 (9-21) 14 (9-21) 14 (10-21) Selective/superselective TACE (n) Serum creatinine (mg/dl) 0.90 ( ) 0.96 ( ) 0.90 ( ) 0.91 ( ) Serum bilirubin (mg/dl) 2.01 ( ) 2.53 ( ) 3.01 ( ) 2.45 ( ) International normalized ratio 1.37 ( ) 1.45 ( ) 1.45 ( ) 1.43 ( ) Model for End-Stage Liver Disease score 13 (7-26) 14 (10-26) 15 (8-27) 14 (9-27) Continuous variables are reported as medians and ranges. Data for all patients include two cases with a combination of the two techniques. The data for day 5 pertain to fewer patients because only those not recovering fast enough were still in the hospital. Those doing well had already been discharged. This might have led to an overestimation of the worsening of liver function. and the stage, an analysis of the histological outcome was carried out for each individual nodule. At the beginning of the observation period, 122 nodules were identified; 53.3% (65 cases) were treated with selective/superselective TACE, whereas the remaining 46.7% were treated with a lobar procedure (57 cases). The characteristics of the treated nodules with respect to the adopted procedures are reported in Table 4; the diameters of the nodules treated with selective/superselective and lobar TACE were similar (P ¼ 0.725), but as expected, multiple tumors were more frequently treated with lobar TACE (P ¼ 0.041). In the explanted liver, the mean treated tumor necrosis level was 64.7%; complete tumor necrosis was obtained in 42.6% (52 cases), whereas the remaining proportion showed different degrees of necrosis. Tumor necrosis was affected by the adopted procedure; it was greater after selective/superselective TACE (75.1%) versus lobar TACE (52.8%, P ¼ 0.002) whether all the nodules were considered as a whole or the nodules were subgrouped according to their size (Table 4). Complete necrosis and necrosis 90% were more frequently observed after selective/superselective TACE versus lobar TACE (P ¼ and P ¼ 0.008, respectively). The treatment of patients with single nodules led to higher levels of tumor necrosis (mean ¼ 86.1%) than the treatment of patients with multiple nodules Table 3. Baseline Clinical and Tumor Characteristics of the Study Population With Respect to the Adopted TACE Procedure TACE Procedure Variable Lobar (n 5 27) Selective/Superselective (n 5 38) P Value Age at start (years) 57 (43-65) 55.5 (37-67) Male gender 25 (92.6%) 35 (92.1%) Serum creatinine (mg/dl) 0.94 ( ) 0.90 ( ) Serum bilirubin (mg/dl) 1.59 ( ) 2.01 ( ) International normalized ratio 1.29 ( ) 1.37 ( ) Child-Pugh class A 22 (81.5%) 30 (78.9%) Model for End-Stage Liver Disease score 12 (8-19) 13 (7-26) Hepatitis B positive serology 7 (25.9%) 9 (23.7%) Hepatitis C positive serology 19 (70.4%) 25 (65.8%) United Network for Organ Sharing T stage at start T1 3 (11.1%) 7 (18.4%) T2 15 (55.6%) 27 (71.1%) T3-T4a 9 (33.3%) 4 (10.5%) Single nodule 10 (37.0%) 22 (57.9%) Number of nodules 2 (1-4) 1 (1-4) Diameter of largest tumor (cm) 3.0 ( ) 2.5 ( ) Sum of tumor diameters (cm) 5.1 ( ) 3.1 ( ) Serum alpha-fetoprotein (ng/ml) 17 (2-735) 10 (2-382) Repeated TACE 16 (59.3%) 12 (31.6%) Continuous variables are reported as median and ranges; other variables are reported as numbers and percentages. The two cases treated with a combination of selective and lobar TACE are not included in this comparison.

7 1586 GOLFIERI ET AL. HEPATOLOGY, May 2011 Table 4. Characteristics of the Nodules in the Explanted Livers With Respect to the Adopted Procedure TACE Procedure Variable All Nodules (122 in 67 Patients) Lobar (57 in 27 Patients) Selective/Superselective (65 in 38 Patients) P Value Degree of necrosis (%) Complete necrosis (100%) 52 (42.6%) 17 (29.8%) 35 (53.8%) Necrosis 90% 65 (53.3%) 23 (40.4%) 43 (66.2%) Necrosis < 50% 40 (32.8%) 25 (43.9%) 15 (23.1%) Diameter of nodules (cm) Number of nodules Single 32 (26.2%) 10 (17.5%) 22 (33.8%) Degree of necrosis (%) Multiple 90 (73.8%) 47 (82.5%) 43 (66.2%) Degree of necrosis (%) Nodule diameter class cm 70 (57.4%) 31 (54.4%) 39 (60.0%) Degree of necrosis (%) cm 31 (25.4%) 13 (22.8%) 18 (27.7%) Degree of necrosis (%) cm 21 (17.2%) 13 (22.8%) 8 (12.3%) Degree of necrosis (%) No other clinical variables were found to be related to the degree of necrosis on pathological examination; variables significantly related to the degree of necrosis were included in the multivariate logistic regression model to identify independent predictors of complete tumor necrosis (see the main text). Continuous variables are reported as means and standard deviations; other variables are reported as numbers and percentages. (57.1%, P ¼ 0.001). The differences between the treatment modalities (selective TACE was better than lobar TACE) were more evident for multiple nodules (P ¼ 0.029; Table 4) than for single nodules (P ¼ 0.172; Table 4). Histological Necrosis According to the Tumor Size. A significant direct relationship between necrosis and the tumor diameter was found, regardless of the type of TACE procedure, in our series of small HCCs: the greater the tumor diameter, the greater the percentage of necrosis. The mean necrosis levels were 59.6% for nodules 2 cm, 68.4% for nodules of 2.1 to 3.0 cm, and 76.2% for nodules > 3.1 cm (P ¼ 0.038; Table 4). As for an analysis of the relationship between the achievement of complete tumor necrosis, the TACE modality, and the tumor diameter, the proportion of patients in each subgroup was too small for a statistically significant comparison to be made. However, because the attainment of complete necrosis resulted from the interaction of the aforementioned variables, a multivariate logistic regression analysis was run: in the study population, independent predictors for achieving complete tumor necrosis were selective/superselective TACE [Exp(B) ¼ 2.192, 95% confidence interval ¼ , P ¼ 0.049] and thetreatmentofasinglenodule[exp(b)¼ 3.756, 95% confidence interval ¼ , P ¼ 0.008]. The nodule diameter played a minor role [Exp(B) ¼ 1.656, 95% confidence interval ¼ , P ¼ 0.089]. Histological Necrosis and Post-TACE CT Assessment. The post-tace CT scan showed homogeneous and dense Lipiodol uptake in all nodules in 44 of 67 patients (65.7%) who were considered complete responders. CT results were considered suspicious for incomplete treatment in 5 patients (7.4%) in whom subsequent CEUS or MRI confirmed viable tumor tissue; in the remaining 18 patients (26.9%), at least one nodule showed incomplete Lipidol uptake on a CT scan. The 44 patients with an apparently complete response were affected by 71 nodules. The 23 patients with suspicious or incomplete Lipiodol uptake had 51 nodules: 24 with complete Lipiodol uptake and 27 with incomplete Lipiodol uptake. In 53 (55.8%) of the 95 nodules with an apparently complete radiological response (dense Lipiodol uptake), complete histological necrosis was confirmed. In all 23 patients with a suspicious or incomplete response, a histological examination confirmed vital tissue. Discussion Taking advantage of the fact that LT offers the possibility of assessing histological tumor necrosis after treatment with TACE, we have been able to show that the possibility of performing a selective/superselective procedure is a highly relevant factor in determining tumor necrosis. At present, TACE is one of the most widely used pre-lt treatments in patients with HCCs. The degree of tumor necrosis induced by TACE has already been reported in the literature, 6,21-29 and there have been different results due to different classifications of the

8 HEPATOLOGY, Vol. 53, No. 5, 2011 GOLFIERI ET AL tumor necrosis rate, different TACE techniques, and, frequently, small sample sizes. Therefore, the effectiveness of TACE in achieving complete tumor necrosis and, consequently, the proper control of tumor progression still has to be clarified. Theoretically, necrosis resulting from treatment provides a beneficial effect by limiting the number of dropouts. The present analysis shows that the main determinant in successful treatment is the adopted procedure modality. In fact, the present data show that the use of selective/superselective TACE leads to the complete necrosis of HCCs approximately 2 times more often than lobar TACE. To the best of our knowledge, at present, only one article 30 has reported a difference between superselective TACE and lobar TACE and their correlation with histological necrosis: the superselective procedure was more often associated with complete tumor necrosis. Interestingly, we also found that patients treated with selective/superselective TACE needed to undergo repeat procedures less frequently. In fact, we repeated TACE only in cases with persistent vital tumors; selective TACE led to a higher rate of complete necrosis and thus limited the need for additional sessions in comparison with whole liver TACE. This might be beneficial in preventing progressive liver and vascular damage. However, we should acknowledge that the present study is not a prospective, randomized study comparing different treatment modalities, and lobar procedures were performed when selective/superselective ones could not be technically carried out for a variety of reasons (but mainly because of the vascular anatomy). It is, therefore, impossible to state with certainty that the results would still have differed if lobar TACE had been performed in patients undergoing selective/superselective TACE, who probably had a more favorable vascular anatomy. Nonetheless, according to the data available in the literature 7 and the current study, we recommend pursuing all technical efforts and attempts to carry out selective/superselective TACE. This statement may appear obvious. However, selective/superselective TACE is usually more time-consuming, more expensive in terms of angiography room occupancy and disposable materials, and more technically demanding than conventional TACE; thus, inexperienced or overloaded operators may be tempted to routinely carry out lobar TACE, which should instead be avoided. We also found that the treatment of single nodules significantly affected tumor necrosis: single nodules showed a higher degree of mean tumor necrosis (86.1%) than multiple nodules (57.1%, P ¼ 0.001). Patients with a single nodule who were treated with selective/superselective or lobar TACE tended to have a higher percentage of necrosis in comparison with patients with multiple nodules who were treated with lobar TACE (P ¼ 0.172). Another interesting finding is the relationship observed between tumor necrosis and the diameter of the nodule. We noted a significantly direct relationship between necrosis and the tumor diameter: the greater the tumor diameter, the greater the percentage of necrosis. To correctly interpret these data, we should consider that our patient population had small HCC nodules (all < 5 cm and almost all < 4 cm). Thus, 21 of the 122 nodules of the complete series (mainly nodules 3-4 cm in size) showed the best response. It is well known that larger HCCs are fed by larger arteries; this leads to better visualization of the nodule during angiographic examination The reverse side of this issue is that smaller nodules (those that have just transitioned from regenerative nodules with severe dysplasia to very early HCCs 34 ) are more often hypovascular; in other words, in these nodules, arterial tumoral neoangiogenesis is not fully developed, and they are still nourished by a limited portal blood supply. 32,34,35 We previously reported 36 that more than 15% of small HCC nodules (1-3 cm) lack the typical contrast HCC pattern at imaging (hyperenhancement in the arterial phase followed by washout), 1,2 and these theoretically correspond to nodules in which vascular derangement has not yet fully taken place; thus, the potential of TACE is limited. In light of these considerations, our finding that the smaller nodules in the present series were less efficiently treated by arterial chemoembolization than the larger ones is not unexpected. Furthermore, Alba and coworkers 12 reported that tumors that were preoperatively detected by CT because of hypervascularity had more necrosis (mean ¼ 67.8%) and were larger (2.58 cm) than those not detected preoperatively (mean necrosis level ¼ 1.57%, diameter ¼ 1.68 cm). Riaz and coworkers 28 found instead that the highest rate of complete necrosis after TACE could be achieved in nodules in the intermediate size range of 3 to 5 cm in comparison with smaller and larger nodules. Our population and consequent findings are different from those patients undergoing TACE as a unique modality (usually with HCCs at an intermediate stage) and their findings. These patients often have nodules > 5 cm and rarely have tumors < 2cm;in this case, an increase in the rate of necrosis in the larger ones would be completely unexpected. Our multivariate logistic regression analysis showed that the independent predictors of complete tumor necrosis were the selective/superselective TACE procedure

9 1588 GOLFIERI ET AL. HEPATOLOGY, May 2011 (P ¼ 0.049) and a single nodule treatment (P ¼ 0.008), whereas the nodule size played a minor role (P ¼ 0.089). In the setting of a locoregional bridge treatment for LT, the rate of complete necrosis according to a pathological analysis after percutaneous radiofrequency ablation was reported to be approximately 50%, but it was 61% to 63% in nodules < 3 cm and 15% in HCCs > 3 cm (usually 3-5 cm). 37 The rate of complete necrosis in the entire series was fully comparable to that achieved after selective/superselective TACE in our present population of transplant patients (53.8%). These data, together with our finding that HCCs 3 to 5 cm in size had a higher rate of necrosis than smaller tumors after TACE, support the strategy (commonly used in many centers) of using a bridge treatment involving percutaneous ablation for smaller nodules (<3 cm) and selective/superselective TACE for larger nodules (>3 cm). Combined treatment might also be an option. As for the post-tace CT assessment, dense Lipidol uptake proved to be a poorly specific marker of a complete histological response. Dedicated trials are warranted to identify the best strategy for post-tace evaluations. In conclusion, the present study shows that in small HCCs (<5 cm), selective/superselective TACE is able to achieve considerable rates of tumor necrosis comparable to the rates reported for HCCs treated with radiofrequency ablation in the same setting of transplant patients; very small nodules (<2 cm), however, do not respond as well as 3- to 4-cm nodules. If selective/superselective procedures cannot be technically performed, lobar procedures may then nonetheless be used, but in this situation, the expected rate of necrosis has been shown to be lower. 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