1
Accepted Article
Article type: Original Article - Hepatology (Clinical) Received date: 27-Feb-2014 Accepted date: 04-Apr-2014 SERUM FERRITIN AS A NEW PROGNOSTIC FACTOR IN
HEPATOCELLULAR CARCINOMA PATIENTS TREATED WITH RADIOFREQUENCY ABLATION 1
Antonio Facciorusso1, Valentina Del Prete1, Matteo Antonino1, Viviana Neve1, Nicola Crucinio1, Alfredo Di Leo2, Brian I Carr3, Michele Barone1
1
Gastroenterology Unit, Department of Medical and Surgical Sciences, University of
Foggia, Ospedali Riuniti Foggia, 2Gastroenterology Unit, Department of Emergency
and Organ Transplantation, University of Bari, Italy, 3Liver Cancer Program, IRCCS de Bellis, Castellana Grotte (BA), Italy
This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/jgh.12618
This article is protected by copyright. All rights reserved.
2
Accepted Article
Correspondence: Prof. Michele Barone Gastroenterology Unit, Department of Medical Sciences University of Foggia AOU Ospedali Riuniti Viale Pinto, 1 71100 Foggia-Italy Fax: 0039 0881733848 Phone: 0039 0881733848 e-mail: [email protected]
Running Title Prognostic role of ferritin after RFA
This article is protected by copyright. All rights reserved.
Accepted Article
3
ABSTRACT
Background and Aim: Hepatic iron accumulation is considered to be a cofactor that influences liver injury and hepatocarcinogenesis. Aim of this study is to determine whether serum ferritin (SF) levels relate to overall survival (OS) and time to recurrence (TTR) in HCC patients treated with percutaneous RFA. Methods:
We measured
SF
levels
in
103
HCC
patients
(median
age
70,
M/F=82.5%/17.5%) who underwent RFA between 2005 and 2010. Correlation between SF and other prognostic factors at baseline was analyzed. SF levels were entered into a Cox
model and their influence on OS and TTR was evaluated in univariate and multivariate analyses. Results:
SF
did
not
correlate
with
alpha-fetoprotein
(rho:
-0.12,
p=0.22),
neutrophil/lymphocyte ratio (rho:-0.1020, p=0.30), MELD (rho:0.18, p=0.06), Child-Pugh score (p=0,5) or BCLC stage (p=0.16). A log-rank test found the value of 244 ng/mL as the optimal prognostic cut-off point for SF.
Median OS was 62 months (54-78) and survival
rate was 97%, 65% and 52% at 1, 4 and 5 years respectively. Performance status and SF were the only predictors of OS at multivariate analysis. Median TTR was 38 months (3449) with a recurrence-free survival rate of 82.5%, 26.2% and 23.3% at 1, 4 and 5 years respectively, while SF and age were the only predictors of TTR. Conclusions: SF level, possibly reflecting the degree of hepatic inflammation and fibrosis, is a negative risk factor for survival and recurrence after percutaneous RFA in HCC patients.
KEY WORDS: HCC; iron; RFA; recurrence, survival
This article is protected by copyright. All rights reserved.
Accepted Article
4
INTRODUCTION Hepatocellular carcinoma (HCC) is a global health problem as it represents the third commonest cause of cancer-related mortality and the leading cause of death among cirrhotic patients.1,2 Current guidelines indicate radiofrequency ablation (RFA) as the standard of care for early HCC patients not suitable to surgery or orthotopic liver transplantation (OLT)2,3. Moreover, several studies published in the last years have showed a competitive effectiveness of RFA as compared to resection for the treatment of single nodules under 2 cm.4,5 Hepatic iron accumulation is considered to be a cofactor that influences liver injury and hepatocarcinogenesis.6 and intrahepatic iron overload, associated with higher levels of
serum ferritin (SF) and iron, has been found to predict a more severe progression of chronic hepatopathy (CH), regardless of its etiology, and a greater risk of HCC development.6 The impact of iron status, and particularly of ferritin levels, on the prognosis of HCC patients’ has been scarcely studied and with conflicting results.7,8 No study has explored the prognostic role of this parameter after a loco-regional therapy such as RFA. SF is an easily measurable, reproducible and cheap test that often forms part of the routine investigations of patients affected from CH.
Aim of this study was to determine whether SF levels influence overall survival (OS) and
time to recurrence (TTR) and therefore can be considered a prognostic factor, in HCC patients treated with percutaneous RFA.
This article is protected by copyright. All rights reserved.
Accepted Article
5
MATERIAL AND METHODS
Patients
From Apr 2005 to Feb 2010, 103 consecutive patients with cirrhosis and a new diagnosis of HCC were treated with percutaneous RFA as a first-line treatment at our Institution. Cirrhosis was diagnosed by histological or clinical features and
liver function was
evaluated according to Child-Pugh (CP) and Model for End-Stage Liver Disease (MELD) score.9 Presence of portal hypertension was defined by at least one of the following:
esophageal varices, platelet count 40,000/ mm3; prothrombin time ratio >40%. SF was dosed by means of electrochemiluminescence immunoassay test (ECLIA®, Roche) with reference range between 25 and 380 ng/mL.
The analysis of the follow up was stopped at December 2013 with a median time of follow up of 78 months (68-82). This article is protected by copyright. All rights reserved.
Accepted Article
6
Treatment protocol RFA was performed under US guidance with a 150 W generator (Model 1500 L; RITA Medical System, Mountain View, California), connected to an expandable 15–14-gauge electrode with a 2.0-cm-long exposed tip (expandable by means of seven hooks). After administration of analgesia (50 to 60 mg of propofol and 0.05 to 0.1 mg of fentanyl) and local anesthesia (5 to 15 mL of 1% lidocaine) by an anesthesiologist, a RFA needle was first inserted into the tumor. The electrode was placed into the center of the lesion maintaining the temperature of the needle tip at 100°C or more for 10–12 min. After ablation, the needle was retracted maintaining its tip hot in order to prevent by thermal coagulation seeding or haemorrhage along the electrode track. For patients with multiple nodules, all lesions were treated in a single session. No antibiotic prophylaxis or anti-
inflammatory drugs were administered prior to therapy. Eventual symptoms occurred after the procedure were managed individually.
Patient monitoring and response evaluation The follow-up protocol included clinical assessment by physical examination, biochemistry, multiphasic CT scan imaging and adverse events (AE) evaluation by means of Common Terminology Criteria for Adverse Events (CTCAE) 4.0.12 at 1 month after the procedure
and, in case of complete response, every 4-6 months. In our study, RFA-related morbidity was defined as any complication within 4 weeks after the procedure. When residual viable tumor was detected by CT-scan in CP ≤B7 patients, a second treatment was planned. Response rate was defined according to modified RECIST (mRECIST) criteria.13 For analytical purpose, in case of consecutive procedures the best response achieved after the last RFA of the treatment series was considered. This article is protected by copyright. All rights reserved.
7
Accepted Article
At recurrence, in case of intrahepatic disease the elective treatment was RFA for single nodules and TACE for multifocal HCC, sorafenib if portal vein thrombosis or metastases had occurred.
Statistical analysis Categorical variables are described as frequencies and percentages and continuous variables as medians and ranges. Correlation between SF and other prognostic factors
was assessed with Spearman’s rank correlation test in case of continuous variables and logistic regression in case of dichotomic parameters. When a logistic regression model was built, goodness of fit was assessed by means of Hosmer–Lemeshow test (HL). Overall survival (OS) and time to recurrence (TTR) were estimated from the date of RFA by Kaplan-Meier with plots and median (95% confidence interval [95% CI]). The inferential analysis for time to event data, namely the factors influencing OS and TTR, was conducted using the Cox univariate and multivariate regression model to estimate hazard ratios (HR) and 95% CI. Statistically significant variables from the univariate Cox analysis were considered for the multivariate models. In order to assess a reliable SF prognostic cut-off value, the running log-rank test was applied.
14
The results obtained with running log-rank statistic for each cutoff point of SF
were plotted against survival and tested for statistical significance via permutations of the data. In brief, on the basis of the cut-off tested, the patients were divided into two groups and compared via log-rank test for each possible cutoff point up to the level that covered 90% of the patients. The SF level which attained the maximum log-rank statistical value was evaluated as an optimal cutoff point. The analysis was performed using R Statistical Software (Foundation for Statistical Computing, Vienna, Austria) and significance was established at the 0.05 level (twosided). This article is protected by copyright. All rights reserved.
Accepted Article
8
RESULTS
Clinical characteristics of patients Table 1 shows clinical and demographic characteristics of the study population. Median age was 70 years (range 39-86), patients were mostly males (82.5%) and HCV was the predominant etiology of the underlying liver disease (60.1%). Median Body Mass Index (BMI) was 23 (17-36) and more than one third of the patients in the study population were diabetics (38.8%). Alcohol abuse was reported in thirty-two (31%) patients. More
than 80% of patients presented a well-preserved liver function within Child-Pugh (CP) A stage and median MELD score was 8 (6-13). Most patients (74.7%) had BCLC A stage with a median sum of tumor diameters of 32 mm (10-84). Median AFP was 25.9 (1.1-2100) UI/mL and median SF levels were 188.5 ng/mL (5-1035). Overall, 157 RFA treatments were performed with a mean number of procedures/patient of 1.52 (± 0.59).
Tumor response and safety data Objective response (complete response + partial response) was observed in 92.1% of patients. Specifically, 87/103 (84.4%) had a complete response and 8/103 (7.7%) a partial response. Mean number of RFA sessions needed to achieve the objective response was 1.4 (± 0.54) with a median time to response of 3 months (95% CI 2-4). No treatment-related deaths were observed. Within 1 month, 3 patients (2.9%) experienced an episode of transient liver decompensation and 3 (2.9%) a severe adverse event (AE grade 3/4), including one case of abdominal abscess and two cases of abdominal pain, from which patients recovered after a brief hospitalization.
This article is protected by copyright. All rights reserved.
9
Accepted Article
Prognostic cut-off level of serum ferritin In order to assess the independence of SF levels from the other markers of inflammation or liver disease, regression models correlating ferritin and the main laboratory and tumoral parameters at baseline were built. Correlation between SF levels and other prognostic factors was analyzed without finding any significances. In fact, SF did not correlate with AFP (rho: -0.12, p=0.22), neutrophil/lymphocyte ratio (NLR [rho:-0.1020, p=0.30]), MELD
(rho:0.18, p=0.06), CP (p=0.5, HL: p=0.32) and BCLC (p=0.16, HL: p=0.27). Interestingly, no correlation between SF levels and BMI (rho:0.004, p=0.16), mellitus diabetes (p=0.73, HL: p=0.45) and alcohol abuse (p=0.74, HL: p=0.52), conditions known as usually associated with hyperferritinemia, was found. In order to define a reliable SF cut-off value able to predict OS, each possible SF level up to the point that covered 90% of the patients was tested via running log-rank analysis. The results are plotted in Figure 1. The most significant cut-off value was 244 ng/mL. Stratifying the study population by this cut-off point, median OS resulted 78 months (59-88) in patients with SF ≤ 244 ng/mL and 31 (24-58) months in patients with SF > 244 ng/mL (HR 2.68 [1.65-4.36], p 244 ng/mL (HR 2.68 [1.65-4.36], p 244 ng/mL (HR 2.36 [1.34-4.16], p 244 ng/mL) were
compared in terms of OS and TTR. In both cases, the group with higher SF levels had a poorer prognosis with shorter survival times and earlier recurrences.
This article is protected by copyright. All rights reserved.
12
Accepted Article
Discordant results have been recently published on the role of SF in predicting outcome in cirrhotic patients on the waiting list for OLT.7,8 However, cirrhotics on the waiting list for OLT represent a population of patients with a more advanced liver disease, in which the outcome is determined by the severity of the underlying hepatopathy, hence in this context it is unlikely that the alteration of a single marker can provide the specificity required for a test to predict important outcome measures. This was supported by the fact that in another study, after controlling for liver prognostic scores, SF failed to predict mortality.7 Furthermore, unlike the present study, only a minor part of the above cited series was made of HCC patients.7,8 In our study, most patients presented with well-preserved liver function (median MELD score 8), hence the aforementioned biases were not found and SF remained as a significant predictor of OS and TTR in both univariate and multivariate Cox test.
Remarkably, we found SF to be a stronger predictor of OS with respect to other recently proposed laboratory indices, such as neutrophil/lymphocyte ratio (NLR). Ferritin may be a more specific marker for hepatic inflammation than NLR and could be related to a more sustained carcinogenetic process in the liver. Interestingly, we found that SF was a strong prognostic factor of earlier recurrence, unlike MELD or CP score. This finding suggests a more specific role of an altered iron status in hepatic carcinogenesis, implying a more sustained rate of local and distant recurrences in patients with higher SF levels . Furthermore, we performed a regression analysis between SF and the main scores of liver function, namely CP and MELD, without finding any significant correlation. This implies that SF is a predictor of RFA outcomes, independently of underlying liver function scores. All the analyses, both correlation and Cox regression, were performed taking into account other possible non liver-related conditions associated with hyperferritinemia, such as BMI, mellitus diabetes and alcohol abuse. In our series, no significant correlations between SF This article is protected by copyright. All rights reserved.
13
Accepted Article
levels and the aforementioned parameters were found and the prognostic role of serum ferritin, both in predicting OS and TTR, was confirmed even when adjustment for these conditions was incorporated in the Cox model. This finding stands for a further promising role of SF in defining HCC prognosis regardless of eventual confounding biases represented by common pathological conditions, such as obesity or diabetes. The main strength of the current study is the novelty of our results that provides a new, reliable and easily measurable prognostic factor for HCC patients. Furthermore, a cut-off point of SF is suggested, thus enabling clinicians to optimize the use of SF as prognostic marker in the routine daily practice. Our paper has some limitations. Firstly, the small number of patients requires further validation by larger multicenter studies. However, only patients with good liver function and preserved performance status were enrolled in our study, reducing the biases for interpretation of the results. Secondly, the low sample of patients with high serum ferritin levels caused a non-linear trend of log HR for OS. Hence, the prognostic cut-off point found in the current study could not be confirmed in broader series presenting a larger range of baseline SF levels. However, our cut-off value could be applicable in patients with less advanced cirrhosis and low tumoral burden, namely the optimal patients for ablative therapies.
In conclusion, our study provides support for the use of new predictors of outcome for HCC patients and can open the way to further investigations concerning the influence of iron-related proteins on HCC prognosis.
This article is protected by copyright. All rights reserved.
Accepted Article
14
DISCLOSURES: The authors declare that they have no conflicts of interest in the research. Author contributions: Valentina Del Prete, Matteo Antonino and Viviana Neve collected the data; Nicola Crucinio performed the technical procedures; Antonio Facciorusso designed the study and performed the statistical analysis; Alfredo Di Leo, Brian I. Carr and Michele Barone reviewed the manuscript. Each author approved the final article and attested to the validity of the results.
This article is protected by copyright. All rights reserved.
15
Accepted Article
REFERENCES 1. El-Serag HB. Current Concepts Hepatocellular Carcinoma. N Engl J Med 2011;365:1118–1127.
2. European Association For The Study Of The Liver, European Organisation For Research And Treatment Of Cancer. EASL-EORTC Clinical Practice Guidelines: Management of hepatocellular carcinoma. J Hepatol 2012;56:908–943.
3. Bruix J, Sherman M. Practice guidelines committee, american association for the study of liver disease. Management of hepatocellular carcinoma. An update. Hepatology 2011;53 (3):1020–1022.
4. Livraghi T, Meloni F, Di Stasi M, et al. Sustained complete response and complications rates after radiofrequency ablation of very early hepatocellular carcinoma in cirrhosis: is resection still the treatment of choice? Hepatology 2008;47:82–89.
5. Pompili M, Saviano A, de Matthaeis N, et al. Long-term effectiveness of resection and radiofrequency ablation for single hepatocellular carcinoma ≤3 cm. Results of a multicenter Italian survey. J Hepatol. 2013;59(1):89-97.
6. Lambrecht RW, Sterling RK, Naishadham D, et al. Iron levels in hepatocytes and portal tract cells predict progression and outcome of patients with advanced chronic hepatitis C. Gastroenterology. 2011; 140(5): 1490–1500.
7. Al-Freah MAB, Kriese S, Foxton MR, et al. The association of pretransplant ferritin level with waiting list and post-transplant survival. Does ferritin actually predict outcome? Transplant International. 2013;26(11):1070-1079.
8. Walker NM, Stuart KA, Ryan RJ, et al. Serum ferritin concentration predicts mortality in patients awaiting liver transplantation. Hepatology 2010; 51: 1683.
9. Kamath PS, Wiesner RH, Malinchoc M, et al: A model to predict survival in patients with end-stage liver disease. Hepatology 2001; 33:464-470
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Accepted Article
10. Mazzaferro V, Sposito C, Bhoori S, et al. Yttrium-90 radioembolization for intermediate-advanced hepatocellular carcinoma: a phase 2 study. Hepatology 2013;57(5):1826-1837.
11. Bruix J, Sherman M. Management of hepatocellular carcinoma. Hepatology 2005;42(5):1208-1236.
12. Common Terminology Criteria for Adverse Events v4.0 NCI, NIH, DHHS. May 29, 2009 NIH publication # 09-7473.
13. Lencioni R, Llovet JM. Modified RECIST (mRECIST) assessment for hepatocellular carcinoma. Semin Liver Dis. 2010;30(1):52-60.
14. Crowley J, LeBlanc M, Jacobson J and Salmon S. Some exploratory tools for survival analysis. In: Lecture Notes on Statistics. Proceedings of the First Seattle Symposium in Biostatistics: Survival Analysis. New York: Springer, 199-229,1997.
15. Wang J, Pantopoulos K. Regulation of cellular iron metabolism. Biochem J 2011; 434: 365.
16. Bell H, Skinningsrud A, Raknerud N, Try K. Serum ferritin and transferrin saturation in patients with chronic alcoholic and non-alcoholic liver diseases. J Intern Med 1994; 236: 315.
17. Kowdley KV, Belt P, Wilson LA, et al. Serum ferritin is an independent predictor of histologic severity and advanced fibrosis in patients with nonalcoholic fatty liver disease. Hepatology 2012; 55: 77.
18. Ba Q, Hao M, Huang H, et al. Iron deprivation suppresses hepatocellular carcinoma growth in experimental studies. Clin Cancer Res. 2011;17(24):7625-7633.
19. Yamasaki T, Terai S, Sakaida I. Deferoxamine for advanced hepatocellular carcinoma. N Engl J Med. 2011;365(6):576-578.
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17
Accepted Article
Table 1. Patients’ baseline characteristics Variable
Age (years)
Gender (M/F)
BMI
Alcohol Abuse (Y/N)
Mellitus Diabetes (Y/N)
70 (39-86)
85 (82.5%)/18 (17.5%)
23 (17-36)
32(31%)/71(69%)
40(38.8%)/63(61.2%)
Etiology (HCV/HBV/Other)
62(60.1%)/23(22.3%)/18(17.6%)
Portal Hypertension (Y/N)
74(71.8%)/29(28.2%)
Child-Pugh (A/B)
86(83.4%)/17(16.6%)
MELD
Neutrophile/Lymphocyte Ratio
8(6-13)
1.4(0.6-3.8)
Serum ferritin (ng/mL)
188.5 (5-1035)
AFP (UI/mL)
25.9(1.1-2100)
Number of Nodules
1(1-4)
Max Diameter (mm)
30 mm(10-65)
Sum of Diameters (mm)
32 mm(10-84)
BCLC (0/A/B)
ALTSG (I/II/III/IVA)
This article is protected by copyright. All rights reserved.
12(11.6%)/77(74.7%)/14(13.7%)
12(11.6%)/80(77.6%)/10(9.7%)/1(0.9%)
18
58(56.3%)/41(39.8%)/5(3.9%)
OKUDA (I/II)
89 (86.4%)/14(13.7%)
Accepted Article
CLIP (0/1/2)
ECOG Performance Status (0/1)
100(97%)/3(3%)
Values are expressed as median (range) or absolute numbers (percentages) when appropriate BMI, Body Mass Index; HCV, hepatitis C virus; HBV, hepatitis B virus; AFP, alpha-fetoprotein; MELD, Model for End stage Liver Disease; BCLC, Barcelona Cancer of the Liver Clinic; ALTSG, American Liver Tumor Study Group; CLIP, Cancer of the Liver Italian Program; ECOG, Eastern Cooperative Oncology Group.
This article is protected by copyright. All rights reserved.
19
Accepted Article
Table 2. Cox univariate/multivariate regression for overall survival.
Variables
Univariate
Multivariate
Analysis
Analysis
Hazard Ratio
Hazard Ratio p-value
p-value
(CI 95%)
(CI 95%)
Age (reference ≤ 65 years)
1.46 (0.86-2.45)
0.16
Gender (reference F)
2.06 (0.98-4.3)
0.054
BMI (reference ≤ 25)
1.78 (0.99-2.34)
0.09
Alcohol Abuse (reference n)
1.91 (1.01-2.98)
0.06
Mellitus Diabetes (reference n)
1.22 (0.76-1.96)
0.40
Etiology (reference HBV)
HCV: 0.74 (0.42-1.31)
0.3
Other: 1.54 (0.77-3.09)
Child-Pugh (reference A)
5.39 (2.88-10.11)
Accepted Article
Article type: Original Article - Hepatology (Clinical) Received date: 27-Feb-2014 Accepted date: 04-Apr-2014 SERUM FERRITIN AS A NEW PROGNOSTIC FACTOR IN
HEPATOCELLULAR CARCINOMA PATIENTS TREATED WITH RADIOFREQUENCY ABLATION 1
Antonio Facciorusso1, Valentina Del Prete1, Matteo Antonino1, Viviana Neve1, Nicola Crucinio1, Alfredo Di Leo2, Brian I Carr3, Michele Barone1
1
Gastroenterology Unit, Department of Medical and Surgical Sciences, University of
Foggia, Ospedali Riuniti Foggia, 2Gastroenterology Unit, Department of Emergency
and Organ Transplantation, University of Bari, Italy, 3Liver Cancer Program, IRCCS de Bellis, Castellana Grotte (BA), Italy
This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/jgh.12618
This article is protected by copyright. All rights reserved.
2
Accepted Article
Correspondence: Prof. Michele Barone Gastroenterology Unit, Department of Medical Sciences University of Foggia AOU Ospedali Riuniti Viale Pinto, 1 71100 Foggia-Italy Fax: 0039 0881733848 Phone: 0039 0881733848 e-mail: [email protected]
Running Title Prognostic role of ferritin after RFA
This article is protected by copyright. All rights reserved.
Accepted Article
3
ABSTRACT
Background and Aim: Hepatic iron accumulation is considered to be a cofactor that influences liver injury and hepatocarcinogenesis. Aim of this study is to determine whether serum ferritin (SF) levels relate to overall survival (OS) and time to recurrence (TTR) in HCC patients treated with percutaneous RFA. Methods:
We measured
SF
levels
in
103
HCC
patients
(median
age
70,
M/F=82.5%/17.5%) who underwent RFA between 2005 and 2010. Correlation between SF and other prognostic factors at baseline was analyzed. SF levels were entered into a Cox
model and their influence on OS and TTR was evaluated in univariate and multivariate analyses. Results:
SF
did
not
correlate
with
alpha-fetoprotein
(rho:
-0.12,
p=0.22),
neutrophil/lymphocyte ratio (rho:-0.1020, p=0.30), MELD (rho:0.18, p=0.06), Child-Pugh score (p=0,5) or BCLC stage (p=0.16). A log-rank test found the value of 244 ng/mL as the optimal prognostic cut-off point for SF.
Median OS was 62 months (54-78) and survival
rate was 97%, 65% and 52% at 1, 4 and 5 years respectively. Performance status and SF were the only predictors of OS at multivariate analysis. Median TTR was 38 months (3449) with a recurrence-free survival rate of 82.5%, 26.2% and 23.3% at 1, 4 and 5 years respectively, while SF and age were the only predictors of TTR. Conclusions: SF level, possibly reflecting the degree of hepatic inflammation and fibrosis, is a negative risk factor for survival and recurrence after percutaneous RFA in HCC patients.
KEY WORDS: HCC; iron; RFA; recurrence, survival
This article is protected by copyright. All rights reserved.
Accepted Article
4
INTRODUCTION Hepatocellular carcinoma (HCC) is a global health problem as it represents the third commonest cause of cancer-related mortality and the leading cause of death among cirrhotic patients.1,2 Current guidelines indicate radiofrequency ablation (RFA) as the standard of care for early HCC patients not suitable to surgery or orthotopic liver transplantation (OLT)2,3. Moreover, several studies published in the last years have showed a competitive effectiveness of RFA as compared to resection for the treatment of single nodules under 2 cm.4,5 Hepatic iron accumulation is considered to be a cofactor that influences liver injury and hepatocarcinogenesis.6 and intrahepatic iron overload, associated with higher levels of
serum ferritin (SF) and iron, has been found to predict a more severe progression of chronic hepatopathy (CH), regardless of its etiology, and a greater risk of HCC development.6 The impact of iron status, and particularly of ferritin levels, on the prognosis of HCC patients’ has been scarcely studied and with conflicting results.7,8 No study has explored the prognostic role of this parameter after a loco-regional therapy such as RFA. SF is an easily measurable, reproducible and cheap test that often forms part of the routine investigations of patients affected from CH.
Aim of this study was to determine whether SF levels influence overall survival (OS) and
time to recurrence (TTR) and therefore can be considered a prognostic factor, in HCC patients treated with percutaneous RFA.
This article is protected by copyright. All rights reserved.
Accepted Article
5
MATERIAL AND METHODS
Patients
From Apr 2005 to Feb 2010, 103 consecutive patients with cirrhosis and a new diagnosis of HCC were treated with percutaneous RFA as a first-line treatment at our Institution. Cirrhosis was diagnosed by histological or clinical features and
liver function was
evaluated according to Child-Pugh (CP) and Model for End-Stage Liver Disease (MELD) score.9 Presence of portal hypertension was defined by at least one of the following:
esophageal varices, platelet count 40,000/ mm3; prothrombin time ratio >40%. SF was dosed by means of electrochemiluminescence immunoassay test (ECLIA®, Roche) with reference range between 25 and 380 ng/mL.
The analysis of the follow up was stopped at December 2013 with a median time of follow up of 78 months (68-82). This article is protected by copyright. All rights reserved.
Accepted Article
6
Treatment protocol RFA was performed under US guidance with a 150 W generator (Model 1500 L; RITA Medical System, Mountain View, California), connected to an expandable 15–14-gauge electrode with a 2.0-cm-long exposed tip (expandable by means of seven hooks). After administration of analgesia (50 to 60 mg of propofol and 0.05 to 0.1 mg of fentanyl) and local anesthesia (5 to 15 mL of 1% lidocaine) by an anesthesiologist, a RFA needle was first inserted into the tumor. The electrode was placed into the center of the lesion maintaining the temperature of the needle tip at 100°C or more for 10–12 min. After ablation, the needle was retracted maintaining its tip hot in order to prevent by thermal coagulation seeding or haemorrhage along the electrode track. For patients with multiple nodules, all lesions were treated in a single session. No antibiotic prophylaxis or anti-
inflammatory drugs were administered prior to therapy. Eventual symptoms occurred after the procedure were managed individually.
Patient monitoring and response evaluation The follow-up protocol included clinical assessment by physical examination, biochemistry, multiphasic CT scan imaging and adverse events (AE) evaluation by means of Common Terminology Criteria for Adverse Events (CTCAE) 4.0.12 at 1 month after the procedure
and, in case of complete response, every 4-6 months. In our study, RFA-related morbidity was defined as any complication within 4 weeks after the procedure. When residual viable tumor was detected by CT-scan in CP ≤B7 patients, a second treatment was planned. Response rate was defined according to modified RECIST (mRECIST) criteria.13 For analytical purpose, in case of consecutive procedures the best response achieved after the last RFA of the treatment series was considered. This article is protected by copyright. All rights reserved.
7
Accepted Article
At recurrence, in case of intrahepatic disease the elective treatment was RFA for single nodules and TACE for multifocal HCC, sorafenib if portal vein thrombosis or metastases had occurred.
Statistical analysis Categorical variables are described as frequencies and percentages and continuous variables as medians and ranges. Correlation between SF and other prognostic factors
was assessed with Spearman’s rank correlation test in case of continuous variables and logistic regression in case of dichotomic parameters. When a logistic regression model was built, goodness of fit was assessed by means of Hosmer–Lemeshow test (HL). Overall survival (OS) and time to recurrence (TTR) were estimated from the date of RFA by Kaplan-Meier with plots and median (95% confidence interval [95% CI]). The inferential analysis for time to event data, namely the factors influencing OS and TTR, was conducted using the Cox univariate and multivariate regression model to estimate hazard ratios (HR) and 95% CI. Statistically significant variables from the univariate Cox analysis were considered for the multivariate models. In order to assess a reliable SF prognostic cut-off value, the running log-rank test was applied.
14
The results obtained with running log-rank statistic for each cutoff point of SF
were plotted against survival and tested for statistical significance via permutations of the data. In brief, on the basis of the cut-off tested, the patients were divided into two groups and compared via log-rank test for each possible cutoff point up to the level that covered 90% of the patients. The SF level which attained the maximum log-rank statistical value was evaluated as an optimal cutoff point. The analysis was performed using R Statistical Software (Foundation for Statistical Computing, Vienna, Austria) and significance was established at the 0.05 level (twosided). This article is protected by copyright. All rights reserved.
Accepted Article
8
RESULTS
Clinical characteristics of patients Table 1 shows clinical and demographic characteristics of the study population. Median age was 70 years (range 39-86), patients were mostly males (82.5%) and HCV was the predominant etiology of the underlying liver disease (60.1%). Median Body Mass Index (BMI) was 23 (17-36) and more than one third of the patients in the study population were diabetics (38.8%). Alcohol abuse was reported in thirty-two (31%) patients. More
than 80% of patients presented a well-preserved liver function within Child-Pugh (CP) A stage and median MELD score was 8 (6-13). Most patients (74.7%) had BCLC A stage with a median sum of tumor diameters of 32 mm (10-84). Median AFP was 25.9 (1.1-2100) UI/mL and median SF levels were 188.5 ng/mL (5-1035). Overall, 157 RFA treatments were performed with a mean number of procedures/patient of 1.52 (± 0.59).
Tumor response and safety data Objective response (complete response + partial response) was observed in 92.1% of patients. Specifically, 87/103 (84.4%) had a complete response and 8/103 (7.7%) a partial response. Mean number of RFA sessions needed to achieve the objective response was 1.4 (± 0.54) with a median time to response of 3 months (95% CI 2-4). No treatment-related deaths were observed. Within 1 month, 3 patients (2.9%) experienced an episode of transient liver decompensation and 3 (2.9%) a severe adverse event (AE grade 3/4), including one case of abdominal abscess and two cases of abdominal pain, from which patients recovered after a brief hospitalization.
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Accepted Article
Prognostic cut-off level of serum ferritin In order to assess the independence of SF levels from the other markers of inflammation or liver disease, regression models correlating ferritin and the main laboratory and tumoral parameters at baseline were built. Correlation between SF levels and other prognostic factors was analyzed without finding any significances. In fact, SF did not correlate with AFP (rho: -0.12, p=0.22), neutrophil/lymphocyte ratio (NLR [rho:-0.1020, p=0.30]), MELD
(rho:0.18, p=0.06), CP (p=0.5, HL: p=0.32) and BCLC (p=0.16, HL: p=0.27). Interestingly, no correlation between SF levels and BMI (rho:0.004, p=0.16), mellitus diabetes (p=0.73, HL: p=0.45) and alcohol abuse (p=0.74, HL: p=0.52), conditions known as usually associated with hyperferritinemia, was found. In order to define a reliable SF cut-off value able to predict OS, each possible SF level up to the point that covered 90% of the patients was tested via running log-rank analysis. The results are plotted in Figure 1. The most significant cut-off value was 244 ng/mL. Stratifying the study population by this cut-off point, median OS resulted 78 months (59-88) in patients with SF ≤ 244 ng/mL and 31 (24-58) months in patients with SF > 244 ng/mL (HR 2.68 [1.65-4.36], p 244 ng/mL (HR 2.68 [1.65-4.36], p 244 ng/mL (HR 2.36 [1.34-4.16], p 244 ng/mL) were
compared in terms of OS and TTR. In both cases, the group with higher SF levels had a poorer prognosis with shorter survival times and earlier recurrences.
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Accepted Article
Discordant results have been recently published on the role of SF in predicting outcome in cirrhotic patients on the waiting list for OLT.7,8 However, cirrhotics on the waiting list for OLT represent a population of patients with a more advanced liver disease, in which the outcome is determined by the severity of the underlying hepatopathy, hence in this context it is unlikely that the alteration of a single marker can provide the specificity required for a test to predict important outcome measures. This was supported by the fact that in another study, after controlling for liver prognostic scores, SF failed to predict mortality.7 Furthermore, unlike the present study, only a minor part of the above cited series was made of HCC patients.7,8 In our study, most patients presented with well-preserved liver function (median MELD score 8), hence the aforementioned biases were not found and SF remained as a significant predictor of OS and TTR in both univariate and multivariate Cox test.
Remarkably, we found SF to be a stronger predictor of OS with respect to other recently proposed laboratory indices, such as neutrophil/lymphocyte ratio (NLR). Ferritin may be a more specific marker for hepatic inflammation than NLR and could be related to a more sustained carcinogenetic process in the liver. Interestingly, we found that SF was a strong prognostic factor of earlier recurrence, unlike MELD or CP score. This finding suggests a more specific role of an altered iron status in hepatic carcinogenesis, implying a more sustained rate of local and distant recurrences in patients with higher SF levels . Furthermore, we performed a regression analysis between SF and the main scores of liver function, namely CP and MELD, without finding any significant correlation. This implies that SF is a predictor of RFA outcomes, independently of underlying liver function scores. All the analyses, both correlation and Cox regression, were performed taking into account other possible non liver-related conditions associated with hyperferritinemia, such as BMI, mellitus diabetes and alcohol abuse. In our series, no significant correlations between SF This article is protected by copyright. All rights reserved.
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Accepted Article
levels and the aforementioned parameters were found and the prognostic role of serum ferritin, both in predicting OS and TTR, was confirmed even when adjustment for these conditions was incorporated in the Cox model. This finding stands for a further promising role of SF in defining HCC prognosis regardless of eventual confounding biases represented by common pathological conditions, such as obesity or diabetes. The main strength of the current study is the novelty of our results that provides a new, reliable and easily measurable prognostic factor for HCC patients. Furthermore, a cut-off point of SF is suggested, thus enabling clinicians to optimize the use of SF as prognostic marker in the routine daily practice. Our paper has some limitations. Firstly, the small number of patients requires further validation by larger multicenter studies. However, only patients with good liver function and preserved performance status were enrolled in our study, reducing the biases for interpretation of the results. Secondly, the low sample of patients with high serum ferritin levels caused a non-linear trend of log HR for OS. Hence, the prognostic cut-off point found in the current study could not be confirmed in broader series presenting a larger range of baseline SF levels. However, our cut-off value could be applicable in patients with less advanced cirrhosis and low tumoral burden, namely the optimal patients for ablative therapies.
In conclusion, our study provides support for the use of new predictors of outcome for HCC patients and can open the way to further investigations concerning the influence of iron-related proteins on HCC prognosis.
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Accepted Article
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DISCLOSURES: The authors declare that they have no conflicts of interest in the research. Author contributions: Valentina Del Prete, Matteo Antonino and Viviana Neve collected the data; Nicola Crucinio performed the technical procedures; Antonio Facciorusso designed the study and performed the statistical analysis; Alfredo Di Leo, Brian I. Carr and Michele Barone reviewed the manuscript. Each author approved the final article and attested to the validity of the results.
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Accepted Article
REFERENCES 1. El-Serag HB. Current Concepts Hepatocellular Carcinoma. N Engl J Med 2011;365:1118–1127.
2. European Association For The Study Of The Liver, European Organisation For Research And Treatment Of Cancer. EASL-EORTC Clinical Practice Guidelines: Management of hepatocellular carcinoma. J Hepatol 2012;56:908–943.
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6. Lambrecht RW, Sterling RK, Naishadham D, et al. Iron levels in hepatocytes and portal tract cells predict progression and outcome of patients with advanced chronic hepatitis C. Gastroenterology. 2011; 140(5): 1490–1500.
7. Al-Freah MAB, Kriese S, Foxton MR, et al. The association of pretransplant ferritin level with waiting list and post-transplant survival. Does ferritin actually predict outcome? Transplant International. 2013;26(11):1070-1079.
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9. Kamath PS, Wiesner RH, Malinchoc M, et al: A model to predict survival in patients with end-stage liver disease. Hepatology 2001; 33:464-470
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10. Mazzaferro V, Sposito C, Bhoori S, et al. Yttrium-90 radioembolization for intermediate-advanced hepatocellular carcinoma: a phase 2 study. Hepatology 2013;57(5):1826-1837.
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Accepted Article
Table 1. Patients’ baseline characteristics Variable
Age (years)
Gender (M/F)
BMI
Alcohol Abuse (Y/N)
Mellitus Diabetes (Y/N)
70 (39-86)
85 (82.5%)/18 (17.5%)
23 (17-36)
32(31%)/71(69%)
40(38.8%)/63(61.2%)
Etiology (HCV/HBV/Other)
62(60.1%)/23(22.3%)/18(17.6%)
Portal Hypertension (Y/N)
74(71.8%)/29(28.2%)
Child-Pugh (A/B)
86(83.4%)/17(16.6%)
MELD
Neutrophile/Lymphocyte Ratio
8(6-13)
1.4(0.6-3.8)
Serum ferritin (ng/mL)
188.5 (5-1035)
AFP (UI/mL)
25.9(1.1-2100)
Number of Nodules
1(1-4)
Max Diameter (mm)
30 mm(10-65)
Sum of Diameters (mm)
32 mm(10-84)
BCLC (0/A/B)
ALTSG (I/II/III/IVA)
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12(11.6%)/77(74.7%)/14(13.7%)
12(11.6%)/80(77.6%)/10(9.7%)/1(0.9%)
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58(56.3%)/41(39.8%)/5(3.9%)
OKUDA (I/II)
89 (86.4%)/14(13.7%)
Accepted Article
CLIP (0/1/2)
ECOG Performance Status (0/1)
100(97%)/3(3%)
Values are expressed as median (range) or absolute numbers (percentages) when appropriate BMI, Body Mass Index; HCV, hepatitis C virus; HBV, hepatitis B virus; AFP, alpha-fetoprotein; MELD, Model for End stage Liver Disease; BCLC, Barcelona Cancer of the Liver Clinic; ALTSG, American Liver Tumor Study Group; CLIP, Cancer of the Liver Italian Program; ECOG, Eastern Cooperative Oncology Group.
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Accepted Article
Table 2. Cox univariate/multivariate regression for overall survival.
Variables
Univariate
Multivariate
Analysis
Analysis
Hazard Ratio
Hazard Ratio p-value
p-value
(CI 95%)
(CI 95%)
Age (reference ≤ 65 years)
1.46 (0.86-2.45)
0.16
Gender (reference F)
2.06 (0.98-4.3)
0.054
BMI (reference ≤ 25)
1.78 (0.99-2.34)
0.09
Alcohol Abuse (reference n)
1.91 (1.01-2.98)
0.06
Mellitus Diabetes (reference n)
1.22 (0.76-1.96)
0.40
Etiology (reference HBV)
HCV: 0.74 (0.42-1.31)
0.3
Other: 1.54 (0.77-3.09)
Child-Pugh (reference A)
5.39 (2.88-10.11)
