Kecheng Xu,M.D., Lizhi Niu,M.D.,Ph.D. Weibing He,M.D., Yisong He,M.D., Jiansheng Zuo,M.D.

ABSTRACT
Purpose.The purpose of this study is to assess the short-term effect of sequential use of percutaneous cryoablation and ethanol ablation in the management of large hepatocellular carcinoma (HCC) (>5 cm).
Subjects and methods.Eighty patients with large HCC tumors (>5 cm in diameter) were enrolled in this study. Therapy consisted of
percutaneous cryoablation followed by repeated ethanol ablation sessions.
Results.Results showed that the volume of tumor necrosis initially induced by cryoablation has been significantly increased after adjuvant ethanol ablation sessions. The complete necrosis was only seen in 56.3% of patients after cryoablation. Following ethanol ablation, ultimately, 88.8% of the patients have attained complete necrosis by the end of the protocol. The difference between the percentages of tumor necrosis before and after the ethanol ablation sessions showed statistical significance (P < 0.001). All the patients with HCC were followed for median 26 months with a range of 4-67 months. There were 30 patients (37.5 %) who showed no evidence of disease, 20 (25%) alive with recurrent HCC, 25 (31.3%) died of the cancer recurrence, and 5 (6.2%) died of causes other than HCC. There were only 4 patients (5.0％) who had local recurrence in the cryoablation sites. The 1-,2-,3-,4-,and 5-year survival rates were 83%, 64%, 58%, 52%, and 43%, respectively. Five-year-disease-free survival rate was 27%.

Conclusion. Sequential use of percutaneous cryoablation and ethanol ablation could result in complete necrosis of large HCC in significantly more patients than percutaneous cryoablation alone, and effectively improve survival of the patients. This protocol of treatment should be useed as the choice for unresectable large HCC.

Key words.hepatocellular carcinoma cryosurgery percutaneous cryoablation ethanol injection

Although surgical resection is the modality of choice for curative treatment of hepatocellular carcinoma (HCC), most patients are not candidates for resection because of underlying hepatic disease and the malignant nature of the tumor[1]. The treatment for large HCC remains to be a challenge.

Cryoablation has proved to be an effective mean for ablation of small HCC as well as controlling large ones [2-5]. Ethanol ablation has been long used as a method of treating small HCC [6-8]. However, for large HCC ethanol ablation has met high failure rate because the areas of necrosis induced by ethanol are too small and ethanol diffuses poorly beyond the tumor that limits obtaining safety margin [9,10]. On the other hand, cryoablation can achieve larger area of tumor necrosis (> 10 cm in diameter) in fewer treatment sessions in comparison with ethanol ablation [11]. The challenge of treatment of large HCC tumors (>10 cm) requires combination of techniques in order to achieve maximal therapeutic effectiveness and to minimize the side effects [12,13].

It is hypothesized that cryoablation followed by ethanol ablation would increase the volume of tumor necrosis due to their different but theoretically synergetic mechanisms (chemical and freezing effects). This study is retrospective analysis of effectiveness of such combination for the treatment of large HCC.

SUBSJECTS AND METHODS

Patients
Eighty HCC patients, including 57 (71.3%) males and 23 (28.7%) females, were enrolled in this study. The median age of the patients was 48 years with a range of 29- and 79- year-old. Out of these 80 patients, 45 (56.3%) were Child’s score A, while the remaining 35 (43.7%) were at score B. All patients had large HCC tumors (>5 cm in diameter) ranging between 5.5 and 14 cm with a mean diameter of 7.8 cm. The tumors’ diameters are listed in Table 1.

Table 1 Tumor size of 80 patients with HCC

The diagnosis of HCC was mainly based on clinical examination, imaging data (ultrasound, CT/MRI, and angiography) and alpha-fetoprotein (AFP). There were 66 cases whose diagnosis was proven by ulltrasound-guided tru-cut biopsy. All patients were carefully evaluated clinically and considered to be poor candidates for conventional hepatic resection.

Treatment protocol
The protocol consisted of a single cryoablation session including multiple overlapping cryoprobes followed (after 2–3 weeks) by repeated ethanol ablation sessions.

Cryoablation: Percutaneous cryoablation was performed under local anesthesia and guidance of ultrasound and CT. The cryogenerator was (EndoCare,Inc.,Irvine, CA, USA) adapted with multiple cryoprobes of different gauge. After skin disinfection, the cryoprobe was advanced toward to the tumor under US-guidance. The tip of the probe was kept 0.5 cm beyond the tumor margin in its center direction. Selection of probe size was determined based on the tumor size. Generally, multiple cryoprobes were placed in a single lesion in an attempt to cover the largest possible volume of the tumor. Once the probe was positioned, freezing was initiated by infusing argon gas, creating a temperature below -160 C at the tip of probe. A freezing phase persisted for 15 minutes. Once the maximal size of the ice ball was reached, thawing was then followed with infusing helium gas. Two freezing-thawing cycles were performed on each lesion, attempting to create a freeze zone covering entire lesion and at least 1cm beyond the margin of the tumor. Finally, the cryoprobe was removed when the tip temperature reached above 0°C, and the resulting tract was sealed off with fibrin glue immediately after removal of the cryoprobe to ensure haemostasis.

Ethanol ablation: Ethanol ablation of the freezing tissue was given at 2-3 weeks after the cryoablation session. a 22- or 23- gauge needle was insertted percutaneously into the lesions under ultrasound guidance and strerile 100% ethanol was then injected into the area. The volume of the ethanol (V) injected was calculated according to the following formula: V = 4/3π(r + 0.5)3 [24], where V is the volume of ethanol to be injected and r is the radius of the tumor. Attempt was made to position the tip of the needle in the portion of the tumor where residual viable tissue was detected based on CT and ultrasound imaging analysis, In each session, 8–12 ml of ethanol were slowly injected over 3–5 min. The needle’s tip was repositioned in order to cover other portions of the tumor. The needle was kept in place for 40 s after completion of injection, and normal saline was then injected during withdrawal of the needle to avoid leakage of ethanol. Injection of ethanol was conducted twice weekly for 2-6 weeks according to the size of residual tumor.

Estimation of response to therapy
The response of treatment was estimated based on standardization criteria suggested by Goldberg [14]. Helical CT of liver was performed three times, i.e., prior to the treatment protocol started, at 2 weeks after the cryoablation and 2 weeks after the ethanol ablation sessions, respectively, in order to assess the degree of tumor necrosis and to define the residual viable tumor segments. After the end of treatment, US-guided fine-needle aspiration was done to confirm tumor necrosis.

On the first CT scan, the index tumor diameter was measured based on the longest axis of the tumor (L1). After the cryoablation session, the ablation was measured along the short axis of non-enhancing part of the tumor (L2). The percentage of ablation zone after cryoablation was calculated as L2/L1×100. After ethanol ablation sessions, the final necrosis was measured based on the same criteria (L3). The percentage of the final necrosis was measured as L3/L1× 100. The results was graded as complete, incomplete and partial response based on more than 90%–100%, 70%–90% and 50%–70% of necrosis, respectively.

Statistical analysis
Survival rate was estimated by the Kaplan-Meier method .Data were analysed using computer with SPSS software (SPSS,Chicago,IL).A p-value of less than 0.05 was considered statistically significant.

RESULTS
All 80 patients underwent single session of cryoablation and repeated ethanol ablations .

Tumor necrosis after cryoablation
Table 2 showed the percentage of tumor necrosis detected in 80 patients with HCC after cryoablation based on CT findings. 56.3% of patients had complete necrosis, 40% incomplete necrosis (70%–90%), and 3.7% partial necrosis (50%–70%). The difference between cryoablation-induced complete necrosis of larger (>7cm) tumor and that of relatively smaller tumors (5–7 cm) was statistically significant (P <0.05).

Table 2 Percentage of necrosis in relation to the original tumor diameter after cryoablationa

a P < 0.05 for those having complete necrosis with tumors <7 and with >7 cm.

Tumor necrosis after ethanol ablation
The ethanol ablation was performed for all the tumors including those which achieved complete necrosis. The volume of injected ethanol varied according to the residual viable tumor volume(Table 3 ).

Table 3 The ethanol injection sessions in the 80 patients

After ethanol ablation ,the percentage of complete tumor necrosis achieved was significantly increased (Table 4 ) when compared with that without ethanol injection. The complete necrosis was only seen in 56.3% of patients after cryoablation. Following ethanol ablation, 88.8% of the patients had complete tumor necrosis by the end of the treatment protocol. The difference between the percentages of tumor necrosis before and that after the ethanol ablation sessions was statistically significant (P < 0.001).

Table 4 Comparison between tumor necrosis after cryoablation alone and after cryoablation followed by ethanol ablation (end of protocol)a

a P < 0.001, regarding the incidence of complete necrosis between
end of protocol and cryoablation

Adverse effects
Adverse effects of cryoablation were listed in Table 5.There was no ablation-associated motality. Common adverse effects were right hypochondial pain and fever which lasted 3-7 days. One patient developed liver capsular cracking and three developed intra-peritoneal bleeding with resolution after conservative treatment. Transient thrombocytopenia occurred in 6 patients within 1-2 weeks following cryoablation, 2 of whom received platelet transfusion. An asymptomatic right-sided pleural effusions occurred in eight patients, whose tumor located in the right lobe close to the dome of the diaphragm. The pleural effusions were resolved spontaneously within 2-3 weeks.

Majority of patients receiving PEI had pain at injection site, fever and a feeling of alcohol intoxication, which were transient and subsided with conservative management. No patient experienced an appreciable risk.

Table 5 Adverse effects of the cryoablation sessions

N.B: more than 1 adverse effect occurred in some patients.

Follow-up and survival
All patients with HCC received follow-up of median 26 months with a range of 4-67 months. There were 30 patients (37.5 %) who showed no evidence of disease,20 (25%) alive with recurrent HCC, 25 (31.3%) died of disease recurrence, and 5 (6.2%) died of causes other than HCC. There were only 4 patients (5.0％) who had the local recurrence at the cryoablation sites. The 1-,2-,3-,4-,and 5-year survival rates were 83%, 64%, 58%, 52%, and 43%, respectively. Five-year-disease-free survival rate was 27%.(Table 6).

Table 6. Survival and disease status after combination treatment

DICUSSION
Hepatic cryotherapy is increasingly recognized as a safe and effective alternative therapy for patients who are not suitable candidates for conventional hepatic resection. Local recurrence after cryoablation has been a main drawback of this therapy, which limits the survival[15]. The frequency of recurrence is influenced by several factors, of which the size of the tumor to be treated has a greater prognostic impact. The larger the lesions are, the higher the recurrence rate is [16].

The higher recurrence rate of the larger tumor is mainly resulted from incomplete destruction in the site of cryoablation[17]. A local recurrence in a cryoablated lesion would be the equivalent of a positive margin on a resected liver specimen, which has been demonstrated to have a negative impact on survival[18]. Temperatures lower than -40 °C are assumed necessary to ensure tumor ablation. Ice-balls larger than the target lesions are necessary for complete tumor ablation, because the temperature at the edge of the iceball is non-lethal. The ice-ball covering entire tumor and 1cm beyond the tumor margin should be considered as adequate for ablation[19]. The larger tumors are often located in the vicinity of large vessels. Blood flowing with a warming effect can result in the insufficient cryodestruction of the tumor, especially for peripheral area of the tumor[20]. Jungraithmayr et al[21]reported that 10 (58.8%) of 17 patients with malignant liver tumor had a relapse at the site of cryolesion after follow-up for an average of 23 months. Among tumors close to venae cava (VC) with large venous affluxes, 58.3% recurred. Pearson et al[22] reported tumors to be treated had 13.6% of local recurrence rate, in which 66.7% occurred directly in the venae cava (VC) or a large vessel. Moreover, To freeze a larger tumor, multiple-cryoprobes are often used, which will result in asymmetric cryoablation.

Ethanol can diffuse into the tumor cells and cause nonselective protein denaturation and cellular dehydration, leading to a coagulated necrosis. Subsequent fibrosis and small vessel thrombosis also contribute to cellular death. Therefore, after cryoablation which could destroy majority of tumors, ethanol injection at incomplete necrosis area of cryoablated tumor can destroy residue tumor tissues.

On the other hand, similar to radiofrequency (RF) ablation[23], cryoablation can strengthen the effect of ethanol ablation: (a) cryoablation ablation intenerates tumor and disruptes the intratumoralsepta , making the residual viable tumor easy to the lethal effect of ethanol ablation; (b) the potential safety margin induced by cryoablation forms a capsule, which retains ethanol inside the tumor and prolongs its effect.

Our results show that the percentage of patients who achieved complete tumor necrosis has jumped from 56.3% after cryoablation alone to 77.5% by the end of the protocol. Saki [17] adopted a protocol of overlapping RF applications, followed by repeated ethanol ablation sessions，for the treatment of large HCC (>5cm) and showed that the percentage of patients who achieved complete tumor necrosis has jumped from 52.5% after RF alone to 80% by the end of the protocol. This figure exceeded that reported by Livraghi [6,7] who treated large hepatic tumors (<9.5 cm in diameter) with RF alone and achieved 90% necrosis in 65.3% and 50%–90% necrosis in 34.7% of their series.

The complete tumor necrosis achieved by combination therapy bring about longer survival and few recurrence at the original cryosite. Our previous series showed the one-year survival rate of 78% in 41 patients with HCC who received cryoablation and were followed for a duration of 14 months in average with a range of 5 to 21 months. In this series, ethanol ablation was given to 36 patients with tumor mass larger than 6 cm in diameter 1-2 weeks after cryoablation. Among the 17 patients who had recurrent tumors, only 3 had recurrence at the original cryosite[24]. Wong[17] performed ultrasound-guided cryoablation for 12 patients with HCC, followed by postoperative ethanol ablation on those patients who were found to have residual tumor or recurrence after the cryoablation procedure. The result showed that 1-and 2-year survival rates were 50% and 20%.There were two patients whose tumors were 8 cm and 13 cm in size, and who had disease-free survival for 3 and 2.5 years, respectively.

Our series showed that, during a follow-up of median 26 months with a range of 4-67 months, 37.5 % of the patients showed no evidence of disease, 25% are alive with recurrent HCC, 31.3% died of disease recurrence, and 6.2% died of causes other than HCC. There were only 5.0％ of patients who had the local recurrence in the cryoablation sites. The 1-,2-,3-,4-,and 5-year survival rates were 83%, 64%, 58%, 52%, and 43%, respectively. Five-year disease-free survival rate was 27%.

The results in this study are comparable to that reported by other authors. Pacella[12] used laser induced thermotherapy (LITT) and transarterial chemoembolization (TACE) and showed that 1, 2 and 3 years local recurrence rate for the main tumor was 7%, respectively. Their cumulative survival rate after 1, 2 and 3 years were 92%, 68% and 40%, respectively. Kamada [25] used TACE followed by ethanol ablation for small advanced HCC. Their 5 years survival rate was 50% compared with the 22% in the group of TACE alone. Bloomston [26] reported that 1 year survival was greater in patients undergoing TACE and RF than TACE alone (100% versus 67%, P = 0.004). However, it is necessary to point out that, in this study, the size of the tumors (>5 cm) treated is larger than that reported by the other authors.

In both techniques, complications encountered were all mild to moderate in severity with no mortality or permanent disability. All the adverse effects were resolved either spontaneously during follow-up or with conservative management. The occurrence rate and the nature of the adverse effects are comparable to those mentioned in the literature [27,28].

In conclusion, sequential use of percutaneous cryoablation and ethanol ablation could result in complete necrosis of large HCC,and effectively improve survival of the patients. This protocol of treatment is proved to be a valuable choice for the unresectable large HCC.

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