Author Affiliations: Center for Liver Cancer, National Cancer Center, 323 Ilsan-ro, Ilsandong-gu, Goyang-si, Gyeonggi-do, Republic of Korea (Kim YK, Kim SH, Lee SD, Lee SA and Park SJ); Department of Surgery, College of Medicine, Kangwon National University, 1 Kangwondaehak-gil, Chuncheon-si, Gangwon-do, 200-701 Republic of Korea (Kim YK)

Corresponding Author: Seong Hoon Kim, MD, PhD, Center for Liver Cancer, National Cancer Center, 323 Ilsan-ro, Ilsandong-gu, Goyang-si, Gyeonggi-do, 410-769, Republic of Korea (Tel: 82-31-9201687; Fax: 82-31- 9201138; Email: kshlj@hanmail.net)

 

© 2014, Hepatobiliary Pancreat Dis Int. All rights reserved.

doi: 10.1016/S1499-3872(14)60251-4

 

Contributors: KYK and KSH designed this study, analyzed the data and wrote the article. KSH, LSA and PSJ performed this study. LSD collected the data and performed this study. KSH is the guarantor.

Funding: None.

Ethical approval: This study was approved by the Institutional Review Board of National Cancer Center, Republic of Korea.

Competing interest: No benefits in any form have been received or will be received from a commercial party related directly or indirectly to the subject of this article.

 

 

BACKGROUND: Preoperative absolute monocyte count in peripheral blood (AMCPB) is closely associated with prognoses in not only various malignancies but also hepatocellular carcinoma (HCC). The purpose of this study was to evaluate whether pretransplant AMCPB predicts posttransplant outcomes in patients with HCC undergoing liver transplantation (LT).

 

METHOD: We retrospectively analyzed relationships between clinicopathologic factors involving pretransplant AMCPB and tumor recurrence or survival in 256 patients who had undergone LT for HCC between January 2005 and April 2012.

 

RESULTS: ROC curve analysis showed that AMCPB >200/mm³ was a risk factor for tumor recurrence; 43 patients showed higher AMCPB (>200/mm³), whereas 213 showed lower AMCPB (≤200/mm³) at the time of LT. On multivariate analysis, pretransplant high AMCPB, positive findings in pretransplant ¹⁸F-FDG PET/CT, pathological maximal tumor size >5 cm, intrahepatic metastasis, moderately or poorly differentiated tumor and microvascular invasion were independent factors affecting recurrence-free survival. When we performed subgroup analysis based on the Milan criteria, high AMCPB was an independent factor for predicting HCC recurrence in patients with tumor beyond the Milan criteria (P=0.004), and not for patients within the criteria.

 

CONCLUSION: This study demonstrated that pretransplant AMCPB could predict tumor recurrence after LT for HCC, especially in patients with tumor beyond the Milan criteria.

 

(Hepatobiliary Pancreat Dis Int 2014;13:250-258)

 

KEY WORDS: liver transplantation; hepatocellular carcinoma; recurrence; survival; monocyte count

Hepatocellular carcinoma (HCC) is one of the main causes of death worldwide. In HCC patients, liver transplantation (LT) is a therapeutic option that offers an opportunity to cure not only tumor, but also the underlying carcinogenic liver disease.^[1] The Milan criteria were introduced by Mazzaferro et al^[1] in 1996 to optimize oncologic outcomes according to the size and number of tumor as assessed by pathomorphological findings. Since then, the criteria have been used for selecting patients who are expected to have 5-year recurrence-free survival (RFS) and overall survival (OS) rates of up to 90% and 70% after LT for HCC, respectively.^[2] Despite excellent results within the Milan criteria for HCC, the risk of recurrence has not yet been completely eliminated in patients who meet the selection criteria.^{[2, 3]} There are still recurrence risks of up to 15%-20%, even though patients fulfill the suitable selection criteria.^{[2, 3]} It is mainly attributable to preoperative radiology that does not assess the two major determinants of cancer recurrence such as poor tumor grade and microvascular invasion.^{[2, 3]} However, routine biopsies for the diagnosis and grading of tumor has been discontinued because of concerns about tumor seeding. Thus, the comprehensive search for surrogate markers of tumor is needed to reflect aggressiveness of HCC.

 

Macrophages have several immunological roles, including antigen processing, cell cytotoxicity, foreign body removal, remodeling of tumor, induction of immunity and modification of inflammation. Recent studies^[4-6] have shown that macrophages are correlated with poor outcomes in various malignancies and taken as a prognostic marker. In HCC cases, preoperative absolute monocyte count in peripheral blood (AMCPB) may be associated with a lower survival rate after diagnosis and poorer prognosis after hepatic resection.^[7] However, it is not clear about clinical value of AMCPB in LT candidates for HCC.

 

The present study was to evaluate whether pretransplant AMCPB could predict posttransplant oncologic outcomes in patients with HCC undergoing LT. Additionally, the role of AMCPB was analyzed based on the Milan criteria.

 

 

Between January 2005 and April 2012, a total of 350 patients underwent LT at the National Cancer Center, Republic of Korea. Two hundred and fifty-eight patients had HCC confirmed by histology. Of these patients, 2 were excluded because of perioperative mortality. The remaining 256 patients were included in the study.

 

When the patients were admitted to our institution for elective LT or emergent LT for acute hepatic failure, complete blood cell (CBC) tests were routinely performed on each patient before LT. When there were several different results of CBC tests, pretransplant AMCPB in this study was defined as the result of AMCPB which was most close to the day of LT. The clinical data of each patient were collected from medical records and were statistically analyzed. All candidates for LT underwent computed tomography or enhanced magnetic resonance imaging and ¹⁸F-fluoro-2-deoxy-D-glucose positron emission tomography/computed tomography (¹⁸F-FDG PET/CT). Alpha-fetoprotein (AFP) levels were also assessed. This study was approved by the Institutional Review Board of National Cancer Center, Republic of Korea.

 

Each explant was examined by a single pathologist and categorized based on tumor number, tumor size, tumor differentiation, and microvascular invasion. The Milan criteria were determined according to the size and number of the tumor. Patients with tumors that were not recognized before transplantation but identified on the explanted liver were regarded as having incidental tumors. For patients with multiple lesions in the liver explants, the major tumor histological grade was recorded. Histopathological criteria for intrahepatic metastasis are based on the concept that metastasis does not result from atypical adenomatous hyperplasia or well-differentiated HCCs. Intrahepatic metastasis was considered "present" if (1) a tumor constituted portal vein thrombi; (2) it grew in contiguity with such thrombi; or (3) it appeared as multiple small nodules around a large poorly differentiated tumor. In contrast, if the initial or recurrent tumor was well differentiated, it was considered multicentric occurrence.^[8]

 

¹⁸F-FDG PET/CT was performed as described previously.^[9] Expert nuclear medicine physician interpreted all of the ¹⁸F-FDG PET/CT images. Positive findings of PET was assessed by the nuclear medicine physician whether the ¹⁸F-FDG uptake in HCC was PET-positive or significantly higher than the surrounding noncancerous tissue of the liver.

 

No adjuvant chemotherapy was administered to any patient after transplantation. Immunosuppressive therapy after LT comprised tacrolimus and steroid and/or mycophenolate mofetil. Steroid was tapered to discontinuation by 3-6 months after LT. A combination of high-dose hepatitis B immunoglobulin (HBIG) and an antiviral agent was used as a prophylactic protocol for hepatitis B virus recurrence after LT. Antiviral agents were given for the first postoperative year after transplantation. Patients were approximately followed up for every 3 months in the first 2 years, and every 6 months thereafter. During the routine follow-up, imaging studies including abdomen CT, chest CT, and bone scan were performed every 3 or 6 months, and AFP level was also assessed. If the recurrence was suspected according to the results of imaging tests, additional PET/CT was performed for the examination of distant metastasis. All hepatic recurrences were initially treated with transarterial chemoembolization (TACE). Subsequent treatment depended on whether the intrahepatic lesion had progressed. Patients with multiple intrahepatic recurrences remained on TACE. If the lesion was deep-seated, local ablation therapy was suggested. Resection was suitable for extrahepatic lesions. If the extrahepatic lesions were unresectable, chemotherapy or radiation was considered. Molecularly targeted anticancer drugs were selectively given to patients with multiple pulmonary metastases.

 

All analyses were performed using SAS version 9.1.3 for Windows (SAS institute, Cary, NC, USA). The optimal cutoff values for high and low AMCPB were selected by the area under receiver operating characteristic (ROC) analysis. Both RFS and OS rates were calculated using the Kaplan-Meier method. The relationship between clinicopathologic characteristics and endpoints were analyzed using Cox regression methods. Multivariate analysis showed that candidate variables were included if the corresponding P value in univariate analysis was less than 0.1. We also made subgroup analyses based on the Milan criteria (beyond the Milan criteria vs within the Milan criteria). Clinicopathologic characteristics were summarized in both high and low AMCPB groups as mean±SD or median values (ranges) for continuous variables and as counts (percentages) for categorical variables. Comparisons were made between the 2 groups using the Chi-square test for categorical variables and Student's t test for continuous variables. A P value less than 0.05 was considered statistically significant.

 

 

The mean age of the patients was 54.0±7.2 years (range 33-74), and 83.2% of the patients were male. The etiologies of cirrhosis included hepatitis B (n=227), hepatitis C (n=14), hepatitis B plus hepatitis C (n=11), and non-B non-C hepatitis (n=4). One hundred and forty-four patients (56.3%) belonged to Child B or C. The mean model for end-stage liver disease (MELD) score of the patients was 14.8±8.2. One hundred and eighty-eight patients (73.4%) had a history of previous treatment for HCC before LT. TACE (n=122) was the most frequent treatment modality. The mean±SD of CBCs was 4096.0±2599.0/mm³ for leukocyte; 80×10³±4.8×10³/mm³ for platelets; 2510.4±2346.7/mm³ for neutrophils; 1042.8±625.9/mm³ for lymphocytes; and 394.0±254.6/mm³ for monocytes. The mean duration between CBC tests and LT was 8.4±8.3 days.

 

To determine the cutoff value affecting HCC recurrence, we used ROC curve analysis based on AMCPB. AMCPB significantly had the area under the ROC curves, which was 0.593 with a 95% confidence interval (CI) for the area between 0.512 and 0.675 for tumor recurrence. When the cutoff point put AMCPB at more than 200/mm³, the sensitivity was 94.5% and the specificity was 19.9%. We defined AMCPB more than 200/mm³ as "high" and AMCPB 200/mm³ or less as "low" in this study.

 

The mean duration of follow-up was 33.6±21.0 months (range 2.2-91.3). The 1-, 3- and 5-year OS rates were 94.3%, 84.3% and 76.2%, respectively and RFS rates were 84.3%, 77.3% and 74.3%, respectively. HCC recurrence or metastasis occurred in 55 patients (21.5%). Of these patients, 39 (70.9%) showed HCC recurrence or metastasis within 1 year and 16 (29.1%) exhibited HCC recurrence or metastasis 1 year after LT. The median time to recurrence was 6.2 months (range 0.8-74.3). A total of 40 patients died of HCC recurrence. The 1-, 3- and 5-year OS rates were 98.2%, 88.9% and 84.6%, respectively, in patients with tumor fulfilling the Milan criteria, whereas they were 85.6%, 70.6%, and 59.9%, respectively in patients with tumor beyond the Milan criteria. The 1-, 3- and 5-year RFS rates were 94.1%, 88.9% and 84.6%, respectively in patients with tumors fulfilling the Milan criteria, whereas the rates were 64.6%, 51.3%, and 51.3%, respectively in patients with tumor beyond the Milan criteria. OS and RFS were significantly higher in patients with tumor fulfilling the Milan criteria than in those with tumor beyond the Milan criteria (P=0.004 for OS and P<0.001 for RFS).

 

Univariate analysis showed OS was significantly shorter in patients with positive findings in pretransplant ¹⁸F-FDG PET/CT, beyond the Milan criteria on pathology, pathological maximal tumor size >5 cm, microvascular invasion, intrahepatic metastasis, moderately or poorly differentiated tumor and monocyte count >200/mm³ (Table 1). Multivariate analysis showed that the positive findings in ¹⁸F-FDG PET/CT (P<0.001, hazard ratio [HR]=3.3, 95% CI=1.6-6.9) and the presence of intrahepatic metastasis (P=0.004, HR=3.4, 95% CI=1.5-7.6) were independent risk factors for OS (Table 2).

 

Univariate analysis revealed that RFS had significant relationships with pretransplant serum AFP levels >200 ng/mL, positive findings in pretransplant ¹⁸F-FDG PET/CT, beyond the Milan criteria on pathology, pathological maximal tumor size >5 cm, pathological multiplicity, microvascular invasion, intrahepatic metastasis, moderately or poorly differentiated tumor, and monocyte count >200/mm³ (Table 1). Multivariate analysis showed that positive findings in ¹⁸F-FDG PET/CT (P=0.001, HR=2.9, 95% CI=1.5-5.5), the presence of intrahepatic metastasis (P<0.001, HR=7.5, 95% CI=3.2-17.6), moderately or poorly differentiated tumor (P=0.016, HR=11.8, 95% CI=1.6-90.0), pathological maximal tumor size >5 cm (P=0.046, HR=1.9, 95% CI=1.0-3.5), and monocyte count >200/mm³ (P=0.038, HR=7.2, 95% CI=1.6-31.7) were independent risk factors for recurrence (Table 2).

 

To identify the prognostic factors especially based on the Milan criteria, subgroup analyses were separately performed in patients with tumor fulfilling or beyond the criteria. In the 97 patients beyond the Milan criteria, multivariate analysis revealed that positive findings in pretransplant ¹⁸F-FDG PET/CT (P=0.001, HR=9.2, 95% CI=2.4-36.2), the presence of microvascular invasion (P=0.024, HR=5.0, 95% CI=1.2-20.4), and the presence of intrahepatic metastasis (P=0.004, HR=18.9, 95% CI=2.5-143.4) were associated with poorer OS. In the 97 patients beyond the Milan criteria, multivariate analysis showed that positive findings in pretransplant ¹⁸F-FDG PET/CT (P=0.011, HR=3.3, 95% CI=1.3-8.4), the presence of microvascular invasion (P=0.020, HR=3.4, 95% CI=1.2-9.5), the presence of intrahepatic metastasis (P<0.001, HR=14.9, 95% CI=3.4-64.5), and AMCPB >200/mm³ (P=0.004, HR=23.8, 95% CI=2.7-210.2) were related to poorer RFS (Fig. 1). In the 159 patients whose HCC met the Milan criteria, multivariate analysis demonstrated that positive findings in pretransplant ¹⁸F-FDG PET/CT (P=0.029, HR=3.1, 95% CI=1.1-8.7) and the presence of intrahepatic metastasis (P=0.007, HR=4.7, 95% CI=1.5-14.4) were related to poorer RFS. Multivariate analysis showed that there is no independent predictor affecting OS in patients with tumor within the Milan criteria. A pretransplant AMCPB >200/mm³ was one of independent predictors affecting recurrence of HCC in patients with tumor beyond the Milan criteria (Fig. 2).

 

Patients with high AMCPB had a higher percentage of larger tumors and a higher frequency of positive findings in pretransplant ¹⁸F-FDG PET/CT. Patients with low AMCPB had significantly lower counts of white blood cells, platelets and neutrophils. This suggested that low pretransplant AMCPB may be significantly related to hypersplenism. There was a strong tendency toward the relationship between poorer liver function (total bilirubin and a higher percentage in Child-Pugh class C) and pretransplant AMCPB; however, it was not statistically significant. Parameters which were related to infection such as acute hepatic failure before LT and isolation of bacteria from blood or ascites were not significantly associated with pretransplant AMCPB.

 

 

This study showed that the pretransplant AMCPB helped to predict tumor recurrence in patients with HCC after LT. Posttransplant RFS rates were significantly poorer in the high AMCPB group than in the low AMCPB group. High pretransplant AMCPB was significantly associated with pathologic tumor size >5 cm and positive findings in pretransplant ¹⁸F-FDG PET/CT in our analysis. Larger HCCs were associated with a higher incidence of vascular invasion and expectedly, with a worse prognosis.^[10] In another large study, HCCs with diameters >4 cm were 3 times more likely to have microvascular invasion than those <4 cm.^[11] These results suggested that larger tumor sizes are also associated with aggressive tumor biology. In addition, reports demonstrated that positive findings in pretransplant ¹⁸F-FDG PET/CT are an independent predictor for tumor recurrence after LT and are closely related to aggressive tumor biology, such as the presence of microvascular invasion and poor differentiation.^[12,13] Our data were in accordance with other studies in the non-transplant settings. Sasaki et al^[7] demonstrated that preoperative AMCPB (>300/mm³) was correlated with tumor aggressiveness as an independent risk factor for recurrence of HCC after resection. These results indicated that patients with high pretransplant AMCPB might have aggressive tumor biology at the time of LT. Thus, HCC might recur more frequently in patients with high pretransplant AMCPB after LT for HCC.

 

Mechanisms underlying the relationship between high AMCPB and decreased survival rate remain unclear. Pollard et al^[14] reported that tumor-associated macrophages (TAMs) were a major component of the inflammatory tumor microenviroment and promoted proliferation and tumor angiogenesis. Kuang et al^{[15, 16]} demonstrated that monocytes were recruited from the circulation into malignant sites or local tissue where they were recognized by residential macrophages. Some of these residential macrophages differentiated into TAMs in response to inflammatory cytokines released by cancer cells. TAMs acted in peritumoral regions as suppressors of the antitumor immune response.^{[15, 16]} In other words, TAMs might be increased in proportion to an increase in AMCPB and suppressed the antitumor immune response. Therefore, patients with high pretransplant AMCPB have more frequent recurrence and poorer survival of HCC.

 

Iwashita et al^[17] reported that tumor immunity could be inhibited by hypersplenism-related liver cirrhosis and HCC. In this study, the low pretransplant AMCPB might be significantly related to hypersplenism. Nevertheless, low pretransplant AMCPB had less frequent recurrence of HCC after LT. This is another evidence to support that AMCPB suppresses antitumor immunity responses rather than antitumor effects.

 

Some reports suggested that patients with poorer liver function had severe inflammatory conditions.^[18-20] Our study showed that there was no significant relationship between poorer liver function and high pretransplant AMCPB. Unlike other inflammatory markers, pretransplant AMCPB could be less affected by liver function.

 

The present study also found that patients with tumor beyond the Milan criteria had poorer RFS when they had high AMCPB at the time of LT. Since most studies about predictive factors for posttransplant recurrence included patients within the Milan criteria, little is known about predictors in patients with tumors beyond the criteria. Patients with tumor beyond the Milan criteria are comparatively heterogeneous in light of both HCC and parameters of liver function compared with those within the criteria who have limited numbers and sizes of tumors. Therefore, it is necessary to suggest special selection criteria for LT in patients beyond the Milan criteria. An et al^[18] found that patients beyond the Milan criteria and with a high level of pretransplant C-reactive protein (1 mg/dL) had poorer OS. The pretransplant neutrophil-lymphocyte ratio predicts tumor recurrence in patients with tumor beyond the Milan criteria.^[21] However, the results of previous studies might be more easily affected by infection or loco-regional treatment. Our unpublished data showed that AMCPB was not associated with loco-regional treatments (P=0.738), such as radiofrequency ablation or TACE. Furthermore, acute hepatic failure before LT and bacterial isolation from blood or ascites were not significantly related to high pretransplant AMCPB. In this study, the measurement of pretransplant AMCPB was an easily conducted and inexpensive method using an extensively available serum marker which could minimize the effects of infection or loco-regional treatment. Therefore, pretransplant AMCPB is a simple but reliable biologic marker for selecting patients when LT is considered in patients with HCC beyond the Milan criteria. Since high pretransplant AMCPB exhibits aggressive tumor biology at the time of LT, one may expect that LT would be performed on patients with high pretransplant AMCPB while considering the possibility of poor outcomes. Paradoxically, LT may be strongly recommended in patients with tumor beyond the Milan criteria and low pretransplant AMCPB.

 

A few prognostic factors were identified in our analysis of patients with HCC fulfilling the Milan criteria. The reasons were possibly as follows: First, the sample size in the present study was relatively small. Second, HCC within the Milan criteria showed a low recurrence probability after LT, which made other variables less significant in this population. Interestingly, positive findings in ¹⁸F-FDG PET/CT were identified as an independent predictor affecting recurrence of HCC in patients with tumors beyond the Milan criteria. Several studies^{[12, 22]} reported that pretransplant ¹⁸F-FDG PET/CT predicted the recurrence and survival in patients who had undergone LT for HCC. Our previous study also indicated that ¹⁸F-FDG-PET/CT predicted early tumor recurrence in HCC patients who had undergone living donor liver transplantation.^[9] In this study, positive findings in ¹⁸F-FDG PET/CT were significantly related to aggressive tumor biologic factors such as intrahepatic metastasis (P<0.001) and microvascular invasion (P=0.013). Therefore, patients beyond the Milan criteria with positive pretransplant findings in ¹⁸F-FDG PET/CT had more frequent recurrence and poorer survival of HCC. Further studies are needed to confirm that pretransplant findings in ¹⁸F-FDG PET/CT would be a predictor in patients with tumors beyond the Milan criteria.

 

This study has some limitations. First, this is a single-institutional retrospective study. Second, there may be other possible factors affecting AMCPB other than infection. However, fastidious examinations were performed to eliminate other clinical problems including infection, and LT was avoided in patients with suspected infection. Even though careful physical examination and laboratory test were performed on LT candidates to exclude those with pretransplant infections and those with intercurrent infections before LT who might have been included in this study. However, the number of such patients was small and the influence was neglectable. Hence, the sample size of this study was relatively small both in patients with HCC beyond and within the Milan criteria. Further prospective studies with larger numbers of patients are required to validate our results.

 

In conclusion, the present study suggested that high pretransplant AMCPB might be a predictor of tumor recurrence in LT candidates for HCC. High AMCPB may exhibit aggressive tumor biology. Additionally, the prognostic value of AMCPB in predicting posttransplant outcomes is higher in patients with tumor beyond the Milan criteria. Therefore, AMCPB may provide valuable information on posttransplant tumor recurrence, especially when selecting LT candidates.

 

 

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