Download

A Concise Review on the Frequency, Major Risk Factors and Surveillance of Hepatocellular Carcinoma (HCC) in β-Thalassemias: Past, Present and Future Perspectives and the ICET-A Experience

Vincenzo De Sanctis1, Ashraf T. Soliman2, Shahina Daar3, Niveen Alansary4, Antonis Kattamis5, Myrto Skafida5, Maria Concetta Galati6, Soteroula Christou7, Saveria Campisi8, Giuseppe Messina9, Mohamed A. Yassin10, Duran Canatan11, Salvatore Di Maio12, Soad Al Jaouni13, Giuseppe Raiola14, Mehran Karimi15, Valeria Kaleva16, Shruti Kakkar17, Demetris Mariannis18 and Christos Kattamis19.

 1 Pediatric and Adolescent Outpatient Clinic, Quisisana Hospital, Ferrara, Italy.
2 Department of Pediatrics, Division of Endocrinology, Hamad General Hospital, Doha, Qatar.
3 Department of Haemtology, College of Medicine & Health Sciences Sultan Qaboos University, Muscat, Oman & Stellenbosch Institute for Advanced Study (STIAS), Wallenberg Research Centre at Stellenbosch University, Stellenbosch, South Africa.
4 Department of Haematology, Sultan Qaboos University Hospital, Sultanate of Oman.
5 Thalassemia Unit, Division of Pediatric Hematology-Oncology. First Department of Pediatrics, University of Athens, “Agia Sofia” Children's Hospital, Athens, Greece.
6 Department of Pediatric Haematoncology, Thalassaemia and Prenatal Diagnosis Regional Center, Pugliese-Ciaccio Hospital, Catanzaro, Italy.
7 Archbishop Makarios III, Thalassemia Center, Nicosia, Cyprus.
8 U.O.S.D. Thalassemia, Umberto 1° Hospital, Siracusa, Italy.
9 SSD Centro per le Microcitemie- Azienda Grande Ospedale Metropolitano, Reggio Calabria,Italy.
10 National Center for Cancer Care and Research, Medical Oncology Hematology Section HMC, Doha, Qatar.
11 Director of Thalassemia Diagnosis Center of Mediterranean Blood Diseases Foundation, Antalya, Turkey.
12 Emeritus Director in Pediatrics, Children’s Hospital “Santobono-Pausilipon”, Naples, Italy.
13 Hematology Department, King Abdulaziz University Hospital, Faculty of Medicine
King Abdulaziz University, Jeddah, Kingdom of Saudi Arabia.
14 Department of Paediatrics, Pugliese-Ciaccio Hospital,Catanzaro, Italy.
15 Hematology Research Center, Shiraz University of Medical Sciences, Shiraz, Iran.
16 Center for Coagulopathies and Rare Anemias,Varna, Bulgaria.
17 Department of Pediatrics, Dayanand Medical College & Hospital, Ludhiana, India.
18 Royal Lancaster Infirmary, United Kingdom.
19 First Department of Pediatrics, Aghia Sophia Children Hospital, National Kapodistrian University of Athens, Athens, Greece.

Corresponding author: Vincenzo De Sanctis MD, Pediatric and Adolescent Outpatient Clinic, Quisisana Hospital, 44100 Ferrara, Italy; Tel.: +39 0532 770243; E-mail: vdesanctis@libero.it

Published: January 1, 2020
Received: October 13, 2019
Accepted: December 3, 2019
Mediterr J Hematol Infect Dis 2020, 12(1): e2020006 DOI 10.4084/MJHID.2020.006

This is an Open Access article distributed under the terms of the Creative Commons Attribution License
(
https://creativecommons.org/licenses/by-nc/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Due to the recent alarming increase in the incidence of hepatocellular carcinoma (HCC) in thalassemias, the present report reviews briefly the frequency, the major risk factors, and the surveillance of HCC in β-thalassemias. Over the past 33 years, 153 cases of HCC were reported in patients with thalassemia, mainly in Italy and Greece. Among HCV-infected patients, additional factors promoting the development of HCC included: advanced age, male sex, chronic hepatitis B (CHB) co-infection, and iron overload. For early diagnosis of HCC, sequential ultrasound screening is recommended especially for thalassemia patients with chronic hepatitis C (CHC), which coincides with (one or more) additional risk factors for HCC. Here we report also the preliminary data from thalassemic patients, above the age of 30 years, followed in 13 ICET-A centers. The total number of enrolled patients was 1,327 (males: 624 and 703 females). The prevalence of HCC in thalassemia major patients [characterized by transfusion-dependency (TDT)] and thalassemia intermedia [characterized by nontransfusion dependency (NTDT)] was 1.66 % and 1.96 % , respectively. The lowest age at diagnosis of HCC was 36 years for TDT and 47 years for NTDT patients. We hope that this review can be used to develop more refined and prospective analyses of HCC magnitude and risk in patients with thalassemia and to define specific international guidelines to support clinicians for early diagnosis and treatment of HCC in thalassemic patients.



Introduction

Hepatocellular carcinoma (HCC) is one of the most common malignant tumors worldwide, representing approximately 90% of all cases of primary liver cancer occuring usually in the setting of chronic liver disease and cirrhosis. Chronic hepatitis C (CHC), chronic hepatitis B (CHB) and hereditary hemochromatosis may directly lead to HCC, while HCC related to other underlying liver diseases is linked to the development of cirrhosis.[1] The global age distribution of HCC varies by region, sex, and aetiology. Rates of liver cancer among persons of the same ethnicity may vary by geographic location.[2-5]
Environmental and acquired individual factors, such as excessive alcohol consumption, fatty liver disease, non-alcoholic steatohepatitis, cigarette smoking, occupational exposure to chemicals such as pesticides, and contamination of foodstuff with aflatoxins (a group of mycotoxins produced by the fungi Aspergillus flavus and Aspergillus parasiticus), can increase the risk of developing liver cancer.[1]
HCC incidence rises after the age of 45 to 50 and becomes almost consistently high after 65 years.[3] HCC is rare among adolescents and accounts for less than 1% of all malignant neoplasms in this age group.[4] The male-to-female ratio in HCC incidence is lower than 2 below 25 years, increases from 25 to 29 years, and peaks at the age of 50-54 years up to 5.40 (95% CI: 5.02, 5.82).[5]
The guidelines for the management of HCC suggested by the American Association for the Study of Liver (AASLD) and the European Association for the Study of Liver (EASL), recommend the implementation of surveillance by a combination of abdominal ultrasound (US), computed tomography (CT) and/or magnetic resonance imaging (MRI). These can improve the prognosis thanks to the detection of smaller tumors.[6,7] Guidelines for HCC in thalassemias are lacking.
The treatment of HCC requires a multidisciplinary approach involving hepatologists, surgeons, oncologists, radiologists, and other disciplines. Multiple options for treatment are available, depending on the tumor stage, patient's performance status and liver function reserve; they include radiofrequency or microwave ablation, liver resection, or transplantation. For patients who are not candidates of curative treatments, locoregional therapies such as trans-arterial chemoembolization (TACE), trans-arterial radio-embolization (TARE), and stereotactic body radiation (SBRT) can improve survival and quality of life. Liver transplantation is another radical therapy for selected patients with HCC.[8]
Sorafenib, a multi-kinase vascular endothelial growth factor (VEGF) inhibitor, is the most widely used systemic chemotherapy approved as a first-line agent for unresectable or advanced HCC.[8]
However, patients with HCC are often diagnosed in advanced stages, and even after complete HCC tumor resection or ablation, the carcinogenic tissue microenvironment in the remnant liver can give rise to recurrent de novo HCC tumors, which progress into incurable, advanced-stage disease in the majority of patients.[9]
The recent alarming increase in the incidence of HCC in thalassemias, prompted us to overview the most common risk factors involved in the development of HCC, to briefly describe the frequency and the clinical and diagnostic characteristics, and to discuss the recommendations for HCC surveillance in high-risk patients with β-thalassemias. The preliminary data of the International Network of Clinicians for Endocrinopathies in Thalassemia and Adolescent Medicine (ICET-A) on HCC survey in thalassemia major (transfusion-dependent thalassemia:TDT) and intermedia patients (transfusion-dependent thalassemia: NTDT) are also presented.

Frequency of HCC in the General Population and in Thalassemias

The global distribution of HCC varies by region, gender, and aetiology. The disease burden is highest in areas with endemic HBV infection (where HBsAg prevalence is 8% or more), such as in sub-Saharan Africa and Eastern Asia, with an HCC incidence rate of over 20 per 100,000 individuals. In Egypt, hepatocellular carcinoma (HCC) is the second most common cancer in men and the 6th most common cancer in women. Italy, Spain, and Greece have intermediate incidence rates of 10-20 per 100,000 individuals, while North and South America have a relatively low incidence (< 5 per 100,000 individuals).[10]
Kourakli et al.[11] analyzed all cases of malignant neoplastic disorders, occurring in 3,652 Greek thalassemic patients, diagnosed between 1985 and 2018. A total of 165 cases of malignant disorders were identified, (84 males and 81 females) with a median age at diagnosis of 45 years (range 9-73 years). The dominant malignancy was hepatocellular carcinoma, diagnosed in 63 patients, followed by thyroid cancer (17 cases), non-Hodgkin's lymphoma (13 cases), and renal cell carcinoma (10 cases). There was a strong positive association between HCV infection and hepatocellular carcinoma.
The literature review showed that HCC in β-thalassemias had been reported in Italy (85 cases), UK (2 cases), Lebanon (2 cases), and Iran (1 case), in addition to Greece (63 cases).[12-23]
In a prospective study, based on ultrasound screening of 105 Italian thalassemic patients, over 18 years of age, Mancuso et al.[16] found a 2% incidence of HCC during a 1-year observation period. Risk factors were present in seventy-two patients: iron overload in 72, HCV infection in 46, HBV infection in 2 and cirrhosis in 10 patients.
Ten years later, Borgna-Pignatti et al. performed a survey of patients followed in 55 Italian centers. The authors collected data from 4,248 patients with thalassemia major and 1,607 patients with thalassemia intermedia. Since their last report,[14] 62 new cases have been identified, of whom 52% with thalassemia major and 45% with thalassemia intermedia and 2 (3%) with compound sickle cell /β- thalassaemia. The cumulative incidence reported at diagnosis of HCC was 1.02% and 1.74%, respectively.[22] The mean age at diagnosis of HCC in patients with TDT was 48 years (33–56 years) and in patients with thalassemia intermedia 47 years (36–72 years); 37 patients (60%) were males and 25 (40%) females.[22]

The ICET-A Experience

In June 2019, to ascertain the frequency of HCC in patients with thalassemias, the Coordinator (VDS) of ICET-A invited 13 centers of the network to take part in a study and collected information on all patients they followed for HCC. An ad hoc questionnaire, prepared by VDS in accordance with the Declaration of Helsinki (http://www.wma.net), was distributed by mail to participating centers. The deadline for sending the requested data was 2 months. The exclusion criteria were: patients with sickle cell disease, and patients already included in other previous publications.
Considering that the youngest patient reported in the literature was 36 years old, we included in the study, only the patients with β-thalassemia above the age of 30 years with β-thalassemias, followed in the participating centers. In detail, the required data were: date of birth, type of haemoglobinopathy, serology for HBV, HCV, detection of HCV-RNA, levels of serum ferritin at diagnosis and chelation therapy, the presence of obesity, alcohol abuse, smoking, and associated clinical complications were also included. In addition, symptoms at onset and clinical course of patients with HCC were reported. Liver iron concentration, measured by magnetic resonance imaging (MRI), was also included.
The demographic details of TDT and NTDT patients, above the age of 30 years, who developed HCC in 13 thalassemia centers from 10 different countries, are presented in table 1.


Table 1 Table 1. Demographic details of TDT and NTDT patients with hepatocellular carcinoma (HCC), above the age of 30 years, in 13 thalassemia centers from 10 different countries.

The total number of enrolled patients with TDT and NTDT was 1,327 (males: 624 and 703 females).
17 patients (mean age: 44.3 years) diagnosed with HCC had TDT (11 males and 6 females), and 6 patients (mean age:56.8 years) had NTDT (5 males and 1 female). The total prevalence of HCC in this subgroup of patients ( > 30 year) was 1.66 % for TDT group and 1.96 % for NTDT. The lowest age at diagnosis of HCC in TDT patients was 36 years and 47 years in NTDT patients, and the highest was 59 and 65, respectively.
The mean age in the group of patients with HCC was higher compared to the patients' group without HCC (TDT and NTDT: 41.0 and 45.7 years vs. 35.5 and 39.2 years, respectively).
Full data was available in 15 out of 23 patients with thalassemias and HCC (Tables 2-4). The presence of obesity, alcohol abuse, or smoking, was absent or very uncommon. The value of αFP at diagnosis was raised ( > 10 ng/mL) in 9 out of 12 patients for whom it was available; 8 out of 14 patients (57.1%) were clinically symptomatic (Tables 2-4). None was positive for HBV surface antigen (HBsAg), while one patient showed evidence of past HBV infection, being HBsAg negative but positive for the antibodies against the HBV core antigen (HBcAb). Interestingly, all Italian patients diagnosed with HCC had genotype 1b. At the time of survey 6 out of 23 patients with thalassemias are alive.


Table 2 Table 2.  Summary of clinical, laboratory and diagnostic data in thalassemic patients with hepatocellular carcinoma (HCC) in Italy.

Table 3 Table 3. Summary of clinical, laboratory and diagnostic data in thalassemic patients with hepatocellular carcinoma (HCC) in Italy, Oman and Greece.

Table 4 Table 4. Summary of clinical, laboratory and diagnostic data in thalassemic patients with hepatocellular carcinoma (HCC) in Cyprus.

Comments. In the general population, the risk for HCC is known to be age dependent; HCC is rarely seen during the first 4 decades of life, except in populations where HBV infection is hyperendemic. The age range at diagnosis of HCC was 63–65 years in Europe and North America, with rare occurrence before the age of 40 years.[3-5]
In our patients with TDT, the mean age at diagnosis of HCC was 44.3 years, younger than that reported in patients without thalassaemia, suggesting the concomitance of other risk factors, namely hemosiderosis, implicated in the development of HCC. Similar results were also reported in 63 Greek thalassemia patients with HCC (median age: 45 years).[11]
Comparing our data with the results of a previous publication,[22] it emerges that our subgroup of NTDT patients with HCC were older (56.8 vs.47 years). However, our series consisted of 6 NTDT patients vs. 28 reported in the multicentre Italian study.[22]
The distribution of HCC varies significantly according to geographic location, and it is more common in middle and low income countries than in developed ones.
It is also interesting to note that no case of HCC in patients with thalassemias was reported in Bulgaria, India, Kingdom of Saudi Arabia, Qatar, (total number of patients:105 patients), and in a large group of patients (292 patients; 125 males and 167 females) followed in a single center in Iran, although the prevalence of HCV Ab and HCV RNA positivity reported in a previous study was 42.8% and 29.9%, respectively.[24] However, the mean age of patients followed in these centers was lower compared to patients with HCC followed in Cyprus, Greece, Italy, and Oman.
In this group of patients with HCC, obesity, NAFLD (assessed by liver ultrasound), or alcoholic abuse was not reported, and NIDDM was diagnosed only in a male TDT patient from Oman (Tables 2-4).
Globally, the rate of males with HCC was higher than of females. This is probably due to two main reasons: (a) the effects of estrogen may suppress the inflammatory process mediated by interleukin-6 (IL-6) in women, reducing hepatic injury and the compensatory proliferation of hepatocytes;[25,26] and (b) the effects of testosterone may increase signalling androgen receptor in men, promoting proliferation of hepatocytes.[27-29]
However, no HCCs have been reported up to now in association with currently used transdermal, subcutaneous, or intramuscular testosterone (T) formulations in hypogonadal thalassemic patients.[30,31]  Nevertheless, because of the virtual "hepatotoxicity” of T preparations, monitoring of liver function is necessary.
On the other hand, the presence of hypogonadism in female thalassemic patients could be a potential additional risk factor of HCC. This hypothesis is supported by a pooled analysis of data from 11 prospective US studies.[32] The authors found that oophorectomy was a significant risk factor for HCC.[32]
However, the role of estrogen-progesterone replacement therapy (HRT) in hypogonadal patients remains controversial. The present evidence suggests both a carcinogenic and protective effect of HRT on liver function.[32-34] Further studies on the indications, duration, and safety of different HRT formulations used in hypogonadal females with thalassemia are required to solve this intriguing puzzle.
In summary, the pivotal role for the evaluation of risk factors of HCC in patients with thalassemias seems to be advanced age, male sex, viral liver infections, and prolonged iron overload.
Further studies on the indications, duration, and safety of different HRT formulations used in hypogonadal patients with thalassemias are required.
Finally, HCC seems to be more common in patients with TDT than NTDT. One possible explanation is that patients with NTDT have a milder progression of iron overloading and a lower incidence of chronic viral liver infections.

Major Risk Factors in Thalassemic Patients with HCC

HCV and HBV. In patients with thalassemias, the predominant role of chronic infection with HBV, and mainly with HCV, in the etiology of HCC is well documented.[12,14,17]
A prospective study to evaluate the incidence and aetiology of transfusion-related hepatitis was reported by Lai et al.[35] Seventy-three of 135 thalassemic patients (62.2%) acquired HCV. An extended follow-up (22 to 30 yr) with HCV RNA assessment was available in 52 patients. Of them, 23 (44.2%) cleared the virus. Fibrosis progression was similar in HCV RNA-positive and HCV RNA-negative patients. Liver iron was the only factor associated with fibrosis.
In another series of 233 HCV positive thalassemic patients, 30% were RNA negative. In these patients, from 2 to 20 years after HCV infection, liver fibrosis was minimal, if any. (Kattamis C et al. unpublished data).
Generally, chronic hepatitis C (CHC) is a slowly progressive disease characterized by persistent hepatic inflammation, leading to the development of cirrhosis in approximately 10-20% of patients over 20-30 years of HCV infection. Overall, once cirrhosis has developed, there is a 1-5% annual risk of HCC and a 3-6% annual risk of hepatic decompensation. The clinical and histopathological disease progress is influenced by several factors, mainly the level of HCV viremia, the virus genotype, the duration of infection, the age at infection, the sex, and the co-existence of active HBV.[36,37]
HCV is classified into 6 major genotypes. Some genotypes have a restricted geographical distribution (genotypes 4-6), while others (genotypes 1-3) are more broadly disseminated. Genotype 1 (subtypes 1a and 1b) is the most prevalent genotype in the world. Genotype 2 is found in clusters in the Mediterranean region; genotype 3 is most prevalent among intravenous drug users and genotype 4 is found mostly in Egypt, while genotypes 5 and 6 are less frequent.[38] The HCV genotypes strongly affect the response to antiviral treatment.
HCV infection is the main risk factor for liver fibrosis in TDT.[39] An accelerated hepatic fibrosis was observed by Kamal et al. in patients with thalassemias and was attributed to the cumulative effect of HCV induced liver injury and the increased iron overload, particularly in inadequately chelated patients.[40]
Efficient chelation therapy may prevent the development of liver fibrosis in thalassemic patients with CHC.[41] Hepatic siderosis and fibrosis were assessed in 99 TDT patients using transient elastography (TE) and liver iron concentration (LIC) at baseline and after 4 years. At baseline, the overall mean liver stiffness measurement (LSM) was 7.4 ± 3.2 kPa, and the mean LIC, assessed by T2*MRI, was 4.81 ± 3.82 mg/Fe g/dw. At 4 ± 1.5 years, a significant reduction in LSM (6.6 ± 3.2 kPa; p: 0.017) and LIC (3.65 ± 3.45 mg/Fe g/dw; p: 0.001) was observed.[42]
Comments. Due to frequent blood transfusion treatment, many patients with thalassemias were infected with HCV, HBV, or both. This applies mostly to patients in their 30s or older, as the risk of viral transmission through blood transfusion was minimized after the 1990s by implementation of blood donor screening in developed and developing countries.
Preventive measures include early immunisation against the hepatitis B virus and prevention of iron accumulation by intensive use of iron chelation therapy. In non-immune thalassemia patients, the implementation of HBV vaccination started in the early 80s. The development of a vaccine for HCV remains an important goal for global control and eradication of infection.
Globally, chronic hepatitis C progresses to liver fibrosis in 60%-70% of patients, cirrhosis in 10%-20%, and eventually HCC in 1%-5% within two decades of harbouring the virus. The ability of HCV to promote cirrhosis is 10- to 20-fold higher than HBV.[3,6-8]
Therefore, patients in the high-risk category should be offered antiviral therapy, as well as appropriate HCC surveillance. Effective suppression of HBV and HCV replication by antiviral therapy can reduce the risk of HCC development. However, antiviral therapy does not eliminate the HCC risk because of the presence of virus integrated into the host genome.
Interferon-based regimens (Peg-IFN) have been the mainstay of anti-HCV therapy, yielding HCV cure, or sustained virologic response (SVR), in approximately 50% of patients, followed by pegylated interferon and ribavirin (Peg-IFN/RBV) for 24 weeks or 48 weeks, the use of which was limited by adverse effects.[43] The host factors influencing response to treatment include: genetics, particularly interleukin (IL)-28B polymorphisms, race, obesity, insulin resistance, severity of hepatic fibrosis, and the related viral genotype and viral load at initiation of therapy.[44]
The recently developed direct-acting antivirals (DAAs), directly targeting the viral protease, polymerase, or non-structural proteins, enabled interferon-free anti-HCV therapies with a revolutionary improvement of SVR rate, approaching or surpassing 90%.[45,46]
The host factors influencing response to treatment include genetics, particularly interleukin (IL)-28B polymorphisms, race, obesity, insulin resistance, the severity of hepatic fibrosis, and the related viral genotype and viral load at initiation of therapy.[46]
The first generation of DAAs, including boceprevir (BOC) and telaprevir (TVR), was introduced in 2011 and added to the previous regimen of pegylated interferon/RBV (Peg IFN/RBV). In October 2014, the FDA approved the use of ledipasvir (LDV) in combination with sofosbuvir (SOF) for the treatment of chronic HCV genotype 1 hepatitis, which had an efficacy of more than 95%. Both drugs are NS3/4A protease inhibitors and were used in combination with peg-interferons and ribavirin to avoid the emergence of resistant variants. These agents improved SVR rates but did not improve the side-effect profile.[47]
Thus, the use of triple therapy with ombitasvir/paritaprevir/dasabuvir, for the treatment of HCV genotype 1 hepatitis, was introduced in December 2014. In the next two years, several other regimes as DAA - sofosbuvir in combination with ledipasvir (NS5A inhibitor), and combination of ombitasvir (NS5A inhibitor), paritaprevir (NS3/4A protease inhibitor), ritonavir (CYP3A inhibitor) and dasabuvir (non-nucleoside NS5B palm polymerase inhibitor), and daclatasvir (NS5A inhibitor) were introduced. The latter was approved for the treatment of HCV genotype 3 infection in combination with sofosbuvir.[47]
In January 2016, the FDA approved the combination treatment of grazoprevir/elbasvir (GZR/EBR) with a 95% SVR.[47] for naïve patients or those who failed treatment with genotype 1 or 4, with or without cirrhosis who failed treatment. The approval of sofosbuvir and velpatasvir (NS5A inhibitor) as a fixed dose combination initiated the third generation of DAAs. However, access to therapies remains limited due to the high costs.[47]
The most extensive observational study on DAAs, in patients with hemoglobinopathies and HCV infection, published to date has been reported by Origa et al.[48]
The majority of patients had TDT (114 patients, 82%), 7 patients (5%) had sickle-cell thalassemia, 3 patients (2%) NTDT and 1 patient (0.7%) sickle-cell anemia. The most common HCV genotype was 1b (91, 65.5%). DAA therapy included different basic and in combination treatment regimens. The mean level of serum ferritin was 1,450 ng/mL (range: 103-11,190 ng/mL) and liver iron overload was observed in 51 (37%) patients. 136 patients (97.8%) achieved a response at the end of treatment, and 130 (93.5%) achieved an SVR. Treatment regimens were well tolerated, and no major adverse events or drug-drug interactions were observed. Treatment with DAAs was also associated with a significant reduction of liver enzymes and serum ferritin.[48]
Supportive evidence for the use of DAAs was reported in 61 thalassemic patients (age range: 40–48 years), including some who previously did not respond to antiviral treatment with pegylated IFN and RBV or/and with advanced liver disease due to CHC. The choice of treatment regimen was mainly based on HCV genotype according to published guidelines and also on the chronological availability of HCV agents. The treatment was highly effective (SVR: 90%) and safe.[49]
In another study, the SVR rate to DAAs in patients with chronic HCV and thalassemia was 82%, while in chronic HCV patients without thalassemia it was higher (94.7%), but not statistically significant.[40] Furthermore, SVR rates were significantly higher in patients who were treated with DAAs compared with PEG-IFN based therapy.[40] Early diagnosis and treatment with DAAs were recommended to prevent cirrhosis and HCC, which are relevant causes of mortality in this patients population.[40]
Iron Overload in TDT and NTDT. The current treatment of TDT consists of regular transfusions to maintain hemoglobin (Hb) levels of 9 to 10 g/dL. Chronic transfusions lead to iron overload and, then, eventual multiorgan damage if not conveniently treated. Therefore, iron chelation therapy is mandatory to remove iron in excess. Regular subcutaneous (SC) desferrioxamine mesylate (DFO) infusion for the treatment of iron overload was started in 1978 in patients older than 2 years, and since 1995 and 2007, two oral chelators, deferiprone (DFP) and deferasirox (DFX) were respectively available and used.
Iron overload is known to cause cancer in animal models,[50] and conversely, iron depletion has been reported to suppress tumor growth.[51]
The first mechanism by which free iron is believed to trigger malignant transformation is the generation of reactive oxygen species (ROS), which causes peroxidation of membrane fatty acids and subsequent formation of toxic by-products that impair protein synthesis and disrupt DNA, leading to mutations in tumor suppressor genes (such as p53) and DNA repair genes.[52,53]
Iron overload may also promote malignant transformation in the liver through the acceleration of fibrosis to cirrhosis by activation of stellate cells and the profibrogenic effects of lipid peroxidation.[54,55]
It also has been suggested that iron overload induces immunologic aberrancies, which may contribute to cancer formation. Growing evidence highlights the negative effect of iron and ferritin on the tumoricidal function of macrophages in mice,[56] in which it was specifically demonstrated that iron overload decreased antibody-mediated and mitogen-stimulated phagocytosis by monocytes and macrophages.[57]
Patients with genetic hemochromatosis were found to be 23 times more likely to have liver cancer than healthy people, with an annual incidence rate of HCC-related cirrhosis of 3%–4%.[58]
Furthermore, a number of case reports and case series have described the development of HCC in patients with thalassemia who were negative for hepatitis B and C testing but had significant hepatic iron overload.[17,21-23]
One NTDT male patient, anti-HCV and HbsAg negative and never transfused, with a previous histological diagnosis of cirrhosis related to secondary hepatic iron overload developed HCC. He was diagnosed at the age of 34 years and was chelated first with deferoxamine and later with deferiprone. Before the diagnosis of HCC, at the age of 73 years, the serum ferritin level was 222 ng/mL.[17] Similar findings were reported by Borgna Pignatti et al.[22] and Maakaron et al.[21,23] in patients with negative serology for HBV and HCV infection.
In a Greek study, HCC was reported in 3 out of 9 NTDT patients with liver fibrosis and siderosis due to the late introduction of iron chelation therapy, and negative history for viral hepatitis.[18] Notably, the incidence of HCC was higher in NTDT compared to TDT (p: 0.032). These findings suggest that the duration of exposure to toxic iron levels may be more important for the development of HCC than the amount of liver iron content.
Comments. Due to frequent and regular blood transfusions, many patients with thalassemia are infected with either HCV or HBV. This occurrence applies mostly to patients in their 30s or older because the risk of viral transmission through blood transfusion was minimized after the 1990s (even annihilated) through blood donor screening.
Preliminary data seem to indicate that NTDT patients might be at a higher risk for developing HCC than TDT patients.[17,20,22] One possible explanation is that patients with NTDT have much longer survival compared to TDT patients, which enables them to live long enough to develop HCC. The state of chronic anemia and hypoxia in NTDT patients, resulting from ineffective erythropoiesis and hemolysis, leads to the expansion of the erythroid marrow and extramedullary hematopoiesis. The chronic ineffective erythropoiesis also triggers increased intestinal iron absorption and deposition in the liver and endocrine glands despite the lack of transfusional iron.[59] Therefore, close surveillance of iron overload via non-invasive quantification of LIC with R2 or T2* MRI is recommended. 

Pathophysiology Past and Recent Issues

Transfusion-Associated Immunomodulation. Increasing evidence suggests that blood transfusions, in addition to iron overload, by inducing chronic antigenic stimulation, have adverse effects on the recipient’s immune system, which is an essential mechanism for antiviral immunity and anticancer immune surveillance.[54,55,58] However, to date, the precise role of transfusion-related immunomodulation in the development of HCC in thalassemia has not yet been elucidated, and needs further studies.[60]
Host genetics factors associated with HCC. Genetic variability has been discussed as a risk factor for the development of HCC since many patients exposed to known environmental risk factors never develop cirrhosis or HCC.[61] Furthermore, family clustering and incidence differences among different ancestry groups suggest that inherited genetic factors may contribute to HCC risk.[62]
From a search of 1,668 publications, Walker et al.[63] identified 166 relevant studies evaluating the associations of HCC with cirrhosis or fibrosis in HCV-infected patients in 137 different genes. IFNL3/4, TNF-α, and PNPLA3 genes had the most probable evidence of association.
In summary, host genetics could add discriminatory value to risk prediction tools, allowing better stratification and personalized assessment of optimal long-term management, thereby increasing the efficacy of surveillance programmes.[63]
Insulin resistance.  Chronic hepatitis C is associated with an increased risk of diabetes mellitus (DM) or insulin resistance (IR).[64,65] IR is associated more frequent in patients with chronic hepatitis C with hepatic steatosis, advanced fibrosis, and HCC.[64] IR may induce the release of free fatty acids (FFA) towards hepatocytes and may cause oxidative stress through the overproduction of ROS, cellular inflammation, and carcinogenesis.
Disturbances of glucose homeostasis, ranging from mild glucose intolerance to overt diabetes mellitus, and hyperinsulinism were reported in young adult patients with thalassemia and have been attributed to iron overload, HCV infection, anemia, and chronic liver disease.[66,67]
An acute effect of blood transfusion on insulin sensitivity and β-cell function in patients with thalassemia has been reported by Wankanit et al.[68]
Alcohol and Tobacco. Alcohol and iron are known prooxidants, and oxidative stress is known to play an essential role in the development of several diseases, including cancer. The metabolism of alcohol, especially through CYP2E1, can lead to the generation of superoxide and hydrogen peroxide. Moreover, hydrogen peroxide can react with ferrous iron (Fe2+) through the Fenton reaction, and generate highly reactive hydroxyl radicals.[69]
Hydroxyl radicals can react with lipid molecules, initiating chain reactions that lead to lipid peroxidation and generation of products, such as acrolein, crotonaldehyde, MDA and 4-HNE; the latter is known to cause mutations of p53 gene (a tumor suppressor gene), which may initiate the development of HCC.[70]
Tobacco exposure is also a risk factor for HCC. Tobacco smoking is associated with increased plasma levels of inflammatory cytokines such as TNF-alpha and IL-1beta[71,72] and markers of oxidative stress.[72,73] These mediators can contribute to necro-inflammatory changes in the liver, which in turn may promote the development of HCC.[74]
In brief, prolonged exposure to alcohol and tobacco is expected to promote the development of HCC in an additive and/or synergistic manner. Tobacco smoking may contribute to the initiation and promotion of HCC due to the presence of mutagenic and carcinogenic compounds as well as by promoting oxidative stress via the generation of ROS and depletion of endogenous antioxidants.
Therefore, thalassemic patients should be discouraged from alcohol consumption and tobacco exposure, regardless of the severity of their disease.
Impact of direct-acting antiviral agents in treated patients. Several retrospective uncontrolled studies in 2016 reported an increased incidence of de novo HCC among patients treated with DAA for HCV infection.[75-77] In a multicentre study of 58 patients in Spain and a single center study of 59 patients in Italy, occurrence or recurrences of HCC after curative therapies in DAA treated patients appeared unusually high (28% and 29%, respectively).[75-77] Although the mechanism underlying this unexpected early HCC recurrence is unknown, it has been reported that HCV eradication by DAA therapy might enhance HCC development or recurrence in patients who have elevated risk for HCC.[78]
In contrast, some other retrospective and prospective cohort studies suggested no significant difference in liver cancer development following DAA therapy.[78-80]
In conclusion, the risk for HCC development is modulated by host features such as gender (male), advanced age, metabolic syndrome, and the severity of the underlying liver disease. The risk for HCC is substantially reduced in patients with CHC who achieve virologic cure, but the risk is not eliminated, and patients should continue surveillance for HCC. Before attempting any interpretation of the discrepancies between the above mentioned DAA reports, long term studies are needed to elucidate this controversial issue.
Vitamin D deficiency. To date, clinical studies have demonstrated that vitamin D deficiency is common, not only in patients with thalassemias,[81] but also in those with HCC. The potential role of vitamin D in HCC has been recently reviewed by Wu et al.[82] and Chiang et al.[83]
Vitamin D is involved in cell proliferation, apoptosis, differentiation, inflammation, invasion and metastasis, angiogenesis and micro-RNA modulation. Although the epidemiologic evidence regarding the association of vitamin D and HCC is still inconclusive, biochemical evidence clearly indicates that HCC cells are responsive to the inhibitory effect of vitamin D and its analogs.[83]
Nonalcoholic fatty liver disease (NAFLD). Metabolic risk factors commonly associated with NAFLD or NASH, including diabetes mellitus type II, obesity, and metabolic syndrome, are becoming emerging risk factors for HCC. The pathophysiology of hepatic carcinogenesis in patients with NAFLD-NASH has not been completely elucidated, but initial research suggests that excess fatty acid supply and hepatocellular steatosis elicit increased fatty acid oxidation with subsequent enhanced reactive oxidative stress. This process further promotes the release of proinflammatory cytokines, prooncogenic signals, and epigenetic changes.[84] Genetic and environmental factors, as well as the interaction between them, may be responsible for both the individual susceptibility and the clinical course of NAFLD.
 

HCC Surveillance

The increasing incidence of HCC in advanced age thalassemic populations led to the need to identify early patients at risk for HCC through an efficient program of screening. Nowadays, almost 50% of HCC are diagnosed at an advanced stage with a poor prognosis and a 5-year overall survival rate lower than 10%. In contrast, in patients with early diagnosis, the 5-year survival rate increases to 70%.[85]
Surveillance consists of the periodic application of a diagnostic test to subjects at risk for developing a specific disease. In one study, the risk factors for HCC in thalassemias were defined as follows: increased serum ferritin (at least 2 determinations > 1,000 ng/ml) or increased LIC >4 mgFe/g dry weight or both; HCV-RNA positivity by polymerase chain reaction and anti-HCV positivity; hepatitis B surface antigen (HBsAg) positivity, or histological diagnosis of cirrhosis.[17]
Currently, the surveillance of HCC in β-thalassemias is achieved by analyzing imaging techniques, abdominal US at 6-12 months frequency, combined with serum α‑fetoprotein (AFP) levels.[15-17,22]
The early detection of HCC by the US is highly dependent on the expertise of the operator and the quality of the equipment. Thus, specialized training for ultrasonographers is recommended. The levels of sensitivity and specificity are also dependent on the size of the tumor, with ultrasound able to detect 80%–95% of tumors 3–5 cm in diameter and 60%–80% of tumors < 1 cm in diameter.[86]
Combining ultrasound and AFP appears to improve the detection rates.[87-89] AFP values may be influenced by non-neoplastic factors, such as viral infections and cirrhosis.[90] At diagnosis of HCC, in thalassemic patients, AFP was within the normal range in 20 out of 45 patients (44.4%) for whom it was available. The median value in TDT patients with HCC was 9.8 µg/L (range 1,1–2,000 µg/L) while in NTDT it was 12.3 µg/L (range:1,5–3,000 µg/L)[22] while in a previous study AFP, at the time of diagnosis was 2,103 ± 2,067 kU/L (normal value <16 kU/L); it was within normal levels in 2 out of 22 patients (9.0%).[14]
The recent introduction of US contrast-enhanced agents (CEUS) has not proven to increase the ability of the US to detect small HCC tumors.[91] However, CEUS provides an accurate differentiation between benign and malignant liver nodules, which is critical for adequate management of HCC and is also useful for guidance of local percutaneous therapy of HCC and post procedure monitoring of the therapeutic response.[92]
Liver fibrosis is a risk factor for HCC, but at what fibrotic stage, the risk for HCC is increased has been poorly investigated. Nakayama et al. reported that a liver stiffness of more than 12.0 kPa was an independent risk factor for HCC development.[93] Therefore, it could be an additional surveillance factor to be included in the surveillance of patients with chronic liver disease and iron overload.
In conclusion, although the optimal methods of screening and the cost-effectiveness of surveillance for HCC remain to be defined, systematic screening still offers the best hope for early diagnosis, treatment eligibility, and improved survival. Current guidelines advocate the use of abdominal US at 6 months frequency to screen for HCC in high-risk patients because the median doubling time of an HCC lesion is 117 days (range 29 to 398 days).[94,95]

Conclusions

A large number of studies have provided evidence that thalassemic patients have a high risk of developing HCC in advanced age. Multicenter international studies for a better evaluation of HCC incidence in different countries are urgently needed. Factors that predispose to HCC, among chronically HCV-infected patients, include advanced age, male sex, viral liver infections, and iron overload (Figure 1). However, several aspects remain to be explored further.

Figure 1 Figure 1. Risk factors for HCC in thalassemias (From: Sciancalepore et al. J Cancer Ther. 2018; 9:417-437; modified).

HCC is more frequently diagnosed (at present) in men with TDT in their fourth - fifth decade and older in NTDT[11,22] with long-standing, untreated chronic hepatitis C (76%), irrespective of the content of hepatic iron load, estimated with MRI T2*.[11] One possible explanation is that patients with NTDT may have a milder process of iron overloading at late age and a lower incidence of chronic viral liver infections, mainly HCV.
HCV is characterized by genetic heterogeneity. Based on the sequence divergence rate, six HCV genotypes and more than 50 subtypes have been identified.[96] The most prevalent HCV subtypes are 1a, 1b, 2a, 2b, and 3a, which are widely distributed globally and account for most HCV infections worldwide.[97] After HCV acute infection an average 50-85% of patients will not clear the virus, and HCV 1b appears to have a high prevalence of HCC.[98]
Interestingly, all 1b HCV genotype-associated neoplasms, reported in the literature, in Greek thalassemic patients were hepatocellular carcinoma (7 cases):[11] a similar pattern was observed in the Italian patients enrolled in our survey.
The youngest thalassemic patient with HCC was 33 years old. Thus at present, we recommend an abdominal US screening associated with AFP levels estimation in all patients over the age of 30 years with an interval of 6 months. For younger patients there are no specific recommendations.
However, we believe that in these patients, surveillance should be based on the evidence of active HBV/HCV infections, iron overload, and advanced liver fibrosis (Figure 2).

Figure 2 Figure 2. Flow chart of HCC surveillance in thalassemias in countries with prolonged implementation of HCV screening of donors.

Liver biopsy has been considered the gold standard of staging liver disease in patients chronically infected with HCV.[99] However, due to its invasive nature, sampling error, the potential for adverse events, and high intra- and interobserver variability,[100] noninvasive diagnostic methods have begun to replace the biopsy in many settings.[7]
Transient elastography (TE; e.g., Fibroscan®), is an accurate and reproducible method to detect liver fibrosis using ultrasound. It is a promising alternative due to its high accuracy in detecting severe fibrosis and cirrhosis.[101]
According to the TE values, patients are grouped into 3 categories: those with elastography values of ≤7.0 kPa corresponding to METAVIR stages F0 or F1 (F0: no fibrosis; F1: portal fibrosis without septa); those with elastography values > 7kPa-≤ 15kPa who have moderate to severe fibrosis (stages F2 and F3) and are at risk for fibrosis progression and the third group includes patients with high elastography values > 15.0 kPa (METAVIR stage of F4 or some cases of F3) who have a high likelihood of cirrhosis.[102]
Noninvasive, serum biomarker panels, which have also been validated against biopsy, are an attractive alternative for staging patients with chronic HCV infection in low- and middle-income countries.[103]
We hope that this review will stimulate the development of more refined, prospective analyses of HCC magnitude and of the risk factors in patients with thalassemia. Finally, we believe that specific international guidelines are urgently needed to support clinicians in the early diagnosis and treatment of thalassemic patients at risk of HCC development.

References   

  1. Shiani A, Narayanan S, Pena L, Friedman M. The Role of Diagnosis and Treatment of Underlying Liver Disease for the Prognosis of Primary Liver Cancer. Cancer Control. 2017 Jul-Sep;24 (3): 10732748 177 29240. https://doi.org/10.1177/1073274817729240 PMid:28975833 PMCid:PMC5937237
  2. Jemal A, Bray F, Center MM, Ferlay J, Ward E, Forman D. Global cancer statistics. CA Cancer J Clin. 2011; 61:69-90. https://doi.org/10.3322/caac.20107 PMid:21296855
  3. Ferenci P, Fried M, Labrecque D, Bruix J, Sherman M, Omata M, Heathcote J, Piratsivuth T, Kew M, Otegbayo JA, Zheng SS, Sarin S, Hamid SS, Modawi SB, Fleig W, Fedail S, Thomson A, Khan A, Malfertheiner P, Lau G, Carillo FJ, Krabshuis J, Le Mair A; World Gastroenterology Organization. World Gastroenterology Organisation guideline. Hepatocellular carcinoma (HCC): a global perspective. J Gastrointestin Liver Dis. 2010;19:311-317. https://doi.org/10.1097/MCG.0b013e3181d46ef2
  4. Darbari A, Sabin KM, Shapiro CN, Schwarz KB. Epidemiology of primary hepatic malignancies in U.S. children. Hepatology. 2003;38:560-566. https://doi.org/10.1053/jhep.2003.50375 PMid:12939582
  5. Liu P, Xie SH, Hu S, Cheng X, Gao T, Zhang C, Song Z. Age-specific sex difference in the incidence of hepatocellular carcinoma in the United States, Oncotarget. 2017 Jul 12;8(40):68131-68137. https://doi.org/10.18632/oncotarget.19245
  6. Bruix, J, Sherman M, American Association for the Study of Liver Diseases. Management of Hepatocellular Carcinoma: An Update. Hepatology.2011;53:1020-1022. https://doi.org/10.1002/hep.24199 PMid:21374666 PMCid:PMC3084991
  7. 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. https://doi.org/10.1016/j.jhep.2011.12.001 PMid:22424438
  8. Mokdad AA, Hester CA, Singal AG, Yopp AC. Management of hepatocellular in the United States. Chin Clin Oncol. 2017 Apr; 6(2):21. https://doi.org/10.21037/cco.2017.04.04 PMid:28482674
  9. Bettinger D, Spode R, Glaser N, Buettner N, Boettler T, Neumann-Haefelin C, Brunner TB, Gkika E, Maruschke L, Thimme R, Schultheiss M. Survival benefit of transarterial chemoembolization in patients with metastatic hepatocellular carcinoma: a single center experience. BMC Gastroenterol. 2017 Aug 10;17(1):98. https://doi.org/10.1186/s12876-017-0656-z PMid:28797231 PMCid:PMC5553671
  10. Mittal S, El-Serag HB. Epidemiology of hepatocellular carcinoma: consider the population, J Clin Gastroenterol. 2013;47 (Suppl):S2-6. https://doi.org/10.1097/MCG.0b013e3182872f29 PMid:23632345 PMCid:PMC3683119
  11. Kourakli A, Diamantidis MD, Skafidas ME, Delicou S, Pantelidou D, Fragodimitri C, Vlachaki E, Lafioniatis S, Petropoulou F, Eftychiadis E, Kapsali E, Kalpaka A, Zissis C, Koutsouka F, Vasileiadi A, Goula A, Giasari P, Katsatou M, Lafiatis I, Klironomos E, Kaiafas P, Kyriacopoulou D, Lazaris V, Maragkos K, Fotiou P, Chalkia P, Schiza V, Kattamis A, Symeonidis A. Hepatitis C Virus Infection, but Not Hepatic Iron Overload Is the Dominant Risk Factor for the Manifestation of Hepatocellular Carcinoma Among Greek Thalassemic Patients. Blood. 2018 132:2347; https://doi.org/10.1182/blood-2018-99-119731
  12. Borgna-Pignatti C, De Stefano P, Sessa F, Avato F. Hepatocellular carcinoma in thalassemia major. Med Pediatr Oncol.1986;14:327-328. https://doi.org/10.1002/mpo.2950140610 PMid:2431257
  13. Zurlo M, De Stefano P, Borgna-Pignatti C, Di Palma A, Piga A, Melevendi C, Di Gregorio F, Burattini MG, Terzoli S. Survival and causes of death in thalassaemia major. Lancet. 1989;334:27-30. https://doi.org/10.1016/S0140-6736(89)90264-X
  14. Borgna-Pignatti C, Vergine G, Lombardo T, Cappellini MD, Cianciulli P, Maggio A, Renda D, Lai ME, Mandas A, Forni G, Piga A, Bisconte MG. Hepatocellular carcinoma in the thalassaemia syndromes. Br J Haematol. 2004;124:114-117. https://doi.org/10.1046/j.1365-2141.2003.04732.x PMid:14675416
  15. Mancuso A, Rigano P, Renda D, Maggio A. Hepatocellular carcinoma on cirrhosis-free liver in a HCV-infected thalassemic. Am J Hematol.2005;78:158-159. https://doi.org/10.1002/ajh.20289 PMid:15682406
  16. Mancuso A, Sciarrino E, Renda MC, Maggio A. A prospective study of hepatocellular carcinoma incidence in thalassemia. Hemoglobin. 2006:30:119-124. https://doi.org/10.1080/03630260500455565 PMid:16540424
  17. Restivo Pantalone G, Renda D, Valenza F, D'Amato F,Vitrano A, Cassarà F, Rigano P, Di Salvo V, Giangreco A, Bevacqua E, Maggio A. Hepatocellular carcinoma in patients with thalassaemia syndromes: Clinical characteristics and outcome in a long term single centre experience. Br J Haematol. 2010;150:245-247. https://doi.org/10.1111/j.1365-2141.2010.08180.x PMid:20433678
  18. Fragatou S, Tsourveloudis I, Manesis G. Incidence of hepatocellular carcinoma in a thalassemia unit. Hemoglobin. 2010: 34:221-226. https://doi.org/10.3109/03630269.2010.485071 PMid:20524812
  19. Ansari S, Azarkivan A, Halagi F. Incidence of hepatocellular carcinoma in patients with thalassemia who had hepatitis C. Acta Med Iran. 2013;51:404-407.
  20. Maakaron JE, Cappellini MD, Graziadei G, Ayache JB, Taher AT. Hepatocellular carcinoma in hepatitis-negative patients with thalassemia intermedia: A closer look at the role of siderosis. Ann Hepatol. 2013; 12: 142-146. https://doi.org/10.1016/S1665-2681(19)31397-3
  21. Modell B, Khan M, Darlison M, Westwood MA, Ingram D, Pennell DJ. Improved survival of thalassaemia major in the UK and relation to T2* cardiovascular magnetic resonance [serial online]. J Cardiovasc Magn Reson. 2008;10:42. https://doi.org/10.1186/1532-429X-10-42 PMid:18817553 PMCid:PMC2563008
  22. Borgna-Pignatti C, Garani MC, Forni GL, Cappellini MD, Cassinerio E, Fidone C, Spadola V, Maggio A, Restivo Pantalone G, Piga A, Longo F, Gamberini MR, Ricchi P, Costantini S, D'Ascola D, Cianciulli P, Lai ME, Carta MP, Ciancio A, Cavalli P, Putti MC, Barella S, Amendola G, Campisi S, Capra M, Caruso V, Colletta G, Volpato S.Hepatocellular carcinoma in thalassaemia: An update of the Italian Registry. Br J Haematol. 2014;167:121-126. https://doi.org/10.1111/bjh.13009 PMid:24992281
  23. Moukhadder HM, Roumi JE, Bou-Fakhredin R, Taher AT. Hepatocellular Carcinoma in a β- Thalassemia Intermedia Patient: Yet Another Case in the Expanding Epidemic. Hemoglobin. 2018; 42:58-60. https://doi.org/10.1080/03630269.2018.1434197 PMid:29493312
  24. Haghpanah S, Jelodari S, Karamifar H, Saki F, Rahimi R, De Sanctis V, Dehbozorgian J, Karimi M. The frequency of hypothyroidism and its relationship with HCV positivity in patients with thalassemia major in southern Iran. Acta Biomed. 2018;89:55-60.
  25. Shi L, Feng Y, Lin H, Ma R, Cai X. Role of estrogen in hepatocellular carcinoma: is inflammation the key? J Transl Med. 2014 Apr 8;12:93. https://doi.org/10.1186/1479-5876-12-93 PMid:24708807 PMCid:PMC3992128
  26. Nagasue N, Ito A, Yukaya H, Ogawa Y. Estrogen receptors in hepatocellular carcinoma. Cancer. 1986;57:87-91. https://doi.org/10.1002/1097-0142(19860101)57:1<87::AID-CNCR2820570118>3.0.CO;2-K
  27. Nagasue N, Ito A, Yukaya H, Ogawa Y. Androgen receptors in hepatocellular carcinoma and surrounding parenchyma. Gastroenterology. 1985;89:643-647. https://doi.org/10.1016/0016-5085(85)90463-9
  28. Pok S, Barn VA, Wong HJ, Blackburn AC, Board P, Farell GC, Teoh NC. Testosterone regulation of cyclin E kinase: a key factor in determining gender differences in hepatocarcinogenesis. J Gastroenterol Hepatol.2016;31:1210-1219. https://doi.org/10.1111/jgh.13232 PMid:26574916
  29. Chen PJ, Yeh SH, Liu WH, Lin CC, Huang HC, Chen CL, Chen DS, Chen PJ. Androgen pathway stimulates microRNA-216a transcription to suppress the tumor suppressor in lung cancer-1 gene in early hepatocarcinogenesis. Hepatology. 2012;56: 632-643. https://doi.org/10.1002/hep.25695 PMid:22392644
  30. De Sanctis V, Elsedfy H, Soliman AT, Elhakim IZ, Pepe A, Kattamis C, Soliman NA, Elalaily R, El Kholy M, Yassin M. Acquired Hypogonadotropic Hypogonadism (AHH) in Thalassaemia Major Patients: An Underdiagnosed Condition? Mediterr J Hematol Infect Dis. 2016 Jan 1;8(1):e2016001. https://doi.org/10.4084/mjhid.2016.001 PMid:26740862 PMCid:PMC4696472
  31. De Sanctis V, Soliman AT, Daar S, Di Maio S. Adverse events during testosterone replacement therapy in 95 young hypogonadal thalassemic men. Acta Biomed. 2019;90:228-232.
  32. Maheshwari S, Sarraj A, Kramer J, El-Serag HB. Oral contraception and the risk of hepatocellular carcinoma. J Hepatol. 2007;47:506-513. https://doi.org/10.1016/j.jhep.2007.03.015 PMid:17462781
  33. McGlynn KA, Sahasrabuddhe VV, Campbell PT, Graubard BI, Chen J, Schwartz LM, Petrick JL, Alavanja MC, Andreotti G, Boggs DA, Buring JE, Chan AT, Freedman ND, Gapstur SM, Hollenbeck AR, Hou L, King LY, Koshiol J, Linet M, Palmer JR, Poynter JN, Purdue M, Robien K, Schairer C, Sesso HD, Sigurdson A, Wactawski-Wende J, Zeleniuch-Jacquotte A. Reproductive factors, exogenous hormone use and risk of hepatocellular carcinoma among US women: results from the Liver Cancer Pooling Project. Br J Cancer. 2015;112:1266-1272. https://doi.org/10.1038/bjc.2015.58 PMid:25742475 PMCid:PMC4385955
  34. De Maria N, Manno M, Villa E. Sex hormones and liver cancer. Mol Cell Endocrinol. 2002;193:59-63. https://doi.org/10.1016/S0303-7207(02)00096-5
  35. Lai ME, Origa R, Danjou F, Leoni GB, Vacquer S, Anni F, Corrias C, Farci P, Congiu G, Galanello R. Natural history of hepatitis C in thalassemia major: a long-term prospective study. Eur J Haematol. 2013;90:501-507. https://doi.org/10.1111/ejh.12086 PMid:23414443
  36. Cho LY, Yang JJ, Ko KP, Park B, Shin A, Lim MK, Oh JK, Park S, Kim YJ, Shin HR, Yoo KY, Park SK. Coinfection of hepatitis B and C viruses and risk of hepatocellular carcinoma: systematic review and meta-analysis. Int J Cancer. 2011;128:176-184. https://doi.org/10.1002/ijc.25321 PMid:20232388
  37. El-Serag HB. Hepatocellular Carcinoma and Hepatitis C in the United States. Hepatology. 2002;36:S74-83. https://doi.org/10.1053/jhep.2002.36807 PMid:12407579
  38. Raimondi S, Bruno S, Mondelli MU, Maisonneuve P. Hepatitis C virus genotype 1b as a risk factor for hepatocellular carcinoma development: a meta-analysis. J Hepatol. 2009;501142-1154. https://doi.org/10.1016/j.jhep.2009.01.019 PMid:19395111
  39. Di Marco V, Capra M, Gagliardotto F, Borsellino Z, Cabibi D, Barbaria F, Ferraro D, Cuccia L, Ruffo GB, Bronte F, Di Stefano R, Almasio PL, Craxì A. et al. Liver disease in chelated transfusion-dependent thalassemics: The role of iron overload and chronic hepatitis C. Hematologica. 2008;93:1243-1246. https://doi.org/10.3324/haematol.12554 PMid:18556410
  40. Kamal S, Abdelhakam S, Ghoraba D, Mohsen MA, Salam AA, Hassan H, Nabeigh L. The Course of Hepatitis C Infection and Response to Anti-viral Therapy in Patients with Thalassemia major and Hepatitis C Infection: A Longitudinal, Prospective Study. Mediterr J Hematol Infect Dis. 2019 Nov 1;11(1): e2019060. https://doi.org/10.4084/mjhid.2019.060 PMid:31700585 PMCid:PMC6827603
  41. Di Marco V, Capra M, Gagliardotto F, Borsellino Z, Cabibi D, Barbaria F, Ferraro D, Cuccia L, Ruffo GB, Bronte F, Di Stefano R, Almasio PL, Craxì A. Liver disease in chelated transfusion-dependent thalassemics: The role of iron overload and chronic hepatitis C. Hematologica. 2008;93:1243-1246. https://doi.org/10.3324/haematol.12554 PMid:18556410
  42. Maira D, Cassinerio E, Marcon A, Mancarella M, Fraquelli M, Pedrotti P, Cappellini MD. Progression of liver fibrosis can be controlled by adequate chelation in transfusion-dependent thalassemia (TDT). Ann Hematol. 2017;96:1931-1936. https://doi.org/10.1007/s00277-017-3120-9 PMid:28875336
  43. Zachou K, Arvaniti P,Gatselis NK, Azariadis K, Papadamou G, Rigopoulou E, Dalekos GN. Patients with haemoglobinopathies and chronic hepatitis C: a real difficult to treat population in 2016? Mediterr J Hematol Infect Dis. 2017, 9(1): e2017003,  https://doi.org/10.4084/mjhid.2017.003 PMid:28101309 PMCid:PMC5224816
  44. Swain MG, Lai MY, Shiffman ML, Cooksley WG, Zeuzem S, Dieterich DT, Abergel A, Pessôa MG, Lin A, Tietz A, Connell EV, Diago M. A sustained virologic response is durable in patients with chronic hepatitis C treated with peg-interferon alfa-2a and ribavirin. Gastroenterology. 2010; 139: 1593-1601. https://doi.org/10.1053/j.gastro.2010.07.009 PMid:20637202
  45. Kohli A, Shaffer A, Sherman A, Kottilil S. Treatment of hepatitis C: a systematic review. J Am Med Assoc 2014;312:631-640. https://doi.org/10.1001/jama.2014.7085 PMid:25117132
  46. Geddawy A, Ibrahim YF, Elbahie NM, Ibrahim MA. Direct acting anti-hepatitis C virus drugs: Clinical pharmacology and future direction. J Transl Intern Med 2017;5:8-17. https://doi.org/10.1515/jtim-2017-0007 PMid:28680834 PMCid:PMC5490957
  47. Zamani F, Ajdarkosh H, Safarnezhad-Tameshkel F, Azarkeivan A, Keyvani H, Naserifar F, Vafaeimanesh J. The effectiveness of sofosbuvir and daclatasvir in the treatment of hepatitis C in thalassaemia major patients and their effect on haematological factors. Indian J Med Microbiol. 2018;36:224-229. https://doi.org/10.4103/ijmm.IJMM_18_90 PMid:30084415
  48. Origa R, Ponti ML, Filosa A, Galeota Lanza A, Piga A, Saracco GM, Pinto V, Picciotto A, Rigano P, Madonia S, Rosso R, D'Ascola D, Cappellini MD, D'Ambrosio R, Tartaglione I, De Franceschi L, Gianesin B, Di Marco V, Forni GL; Italy for THAlassemia and hepatitis C Advance - Società Italiana Talassemie ed Emoglobinopatie (ITHACA-SITE). Treatment of hepatitis C virus infection with direct-acting antiviral drugs is safe and effective in patients with hemoglobinopathies. Am J Hematol. 2017;92:1349-1355. https://doi.org/10.1002/ajh.24911 PMid:28929515
  49. Sinakos E, Kountouras D, Koskinas J, Zachou K, Karatapanis S, Triantos C, Vassiliadis T, Goulis I, Kourakli A, Vlachaki E, Toli B, Tampaki M, Arvaniti P, Tsiaoussis G, Bellou A, Kattamis A, Maragkos K, Petropoulou F, Dalekos GN, Akriviadis E, Papatheodoridis GV. Treatment of chronic hepatitis C with direct-acting antivirals in patients with β-thalassaemia major and advanced liver disease.Br J Haematol. 2017;178:130-136. https://doi.org/10.1111/bjh.14640 PMid:28439915
  50. Okada S, Hamazaki S, Toyokuni S, Midorikawa O. Induction of mesothelioma by intraperitoneal injections of ferric saccharate in male Wistar rats. British J Cancer. 1989; 60:708-711. https://doi.org/10.1038/bjc.1989.344 PMid:2803947 PMCid:PMC2247310
  51. Yu Y, Suryo Rahmanto Y, Richardson DR. Bp44mT: an orally active iron chelator of the thiosemicarbazone class with potent anti-tumour efficacy. British J Pharmacol. 2012; 165:148-166. https://doi.org/10.1111/j.1476-5381.2011.01526.x PMid:21658021 PMCid:PMC3252974
  52. Kew MC. Hepatic iron overload and hepatocellular carcinoma. Cancer Lett.2009;286:38-43. https://doi.org/10.1016/j.canlet.2008.11.001 PMid:19081672
  53. Tirnitz-Parker JE, Glanfield A, Olynyk JK, Ramm GA. Iron and hepatic carcinogenesis. Crit Rev Oncog. 2013; 18:391-407. https://doi.org/10.1615/CritRevOncog.2013007759 PMid:23879586
  54. Kowdley KV. Iron, hemochromatosis, and hepatocellular carcinoma. Gastroenterology. 2004;127 (5 suppl 1):S79-S86. https://doi.org/10.1016/j.gastro.2004.09.019 PMid:15508107
  55. Kew MC. Hepatic iron overload and hepatocellular carcinoma. Liver Cancer 2014; 3:31-40. https://doi.org/10.1159/000343856 PMid:24804175 PMCid:PMC3995380
  56. Matzner Y, Hershko C, Polliack A, Konijn AM, Izak G. Suppressive effect of ferritin on in vitro lymphocyte function. Br J Haematol.1979;42:345-353. https://doi.org/10.1111/j.1365-2141.1979.tb01142.x PMid:157770
  57. Walker EM, Walker SM. Effects of iron overload on the immune system. Ann Clin Lab Sci. 2000; 30:354-365. https://doi.org/10.1023/A:1003905109007
  58. Bruix J, Sherman M. Management of hepatocellular carcinoma: An update. Hepatology. 2011; 53:1020-1022. https://doi.org/10.1002/hep.24199 PMid:21374666 PMCid:PMC3084991
  59. Sleiman J, Tarhini A, Bou-Fakhredin R, Saliba AN, Cappellini MD, Taher AT.Non-Transfusion-Dependent Thalassemia: An Update on Complications and Management. Int J Mol Sci. 2018 Jan 8;19(1). pii: E182. https://doi.org/10.3390/ijms19010182 PMid:29316681 PMCid:PMC5796131
  60. Finianos A, Matar CF, Taher A. Hepatocellular Carcinoma in β-Thalassemia Patients: Review of the Literature with Molecular Insight into Liver Carcinogenesis. Int J Mol Sci. 2018 Dec 17;19(12). pii: E4070. doi: 10.3390/ijms19124070 https://doi.org/10.3390/ijms19124070 PMid:30562917 PMCid:PMC6321074
  61. El-Serag HB, Rudolph KL. Hepatocellular carcinoma: epidemiology and molecular carcinogenesis. Gastroenterology 2007;132:2557-2576. https://doi.org/10.1053/j.gastro.2007.04.061 PMid:17570226
  62. Shen FM, Lee MK, Gong HM, Cai XQ, King MC. Complex segregation analysis of primary hepatocellular carcinoma in Chinese families: interaction of inherited susceptibility and hepatitis B viral infection. Am J Hum Genet.1991;49:88-93.
  63. Walker AJ, Peacock CJ, Pedergnana V; STOP-HCV Consortium, Irving WL. Host genetic factors associated with hepatocellular carcinoma in patients with hepatitis C virus infection: A systematic review. J Viral Hepat. 2018;25:442-456. https://doi.org/10.1111/jvh.12871 PMid:29397014 PMCid:PMC6321980
  64. Raff EJ, Kakati D, Bloomer JR, Shoreibah M, Rasheed K, Singal AK. Diabetes mellitus predicts occurrence of cirrhosis and hepatocellular cancer in alcoholic liver and nonalcoholic fatty liver diseases. J Clin Transl Hepatol. 2015;3:9-16. https://doi.org/10.14218/JCTH.2015.00001 PMid:26356325 PMCid:PMC4542082
  65. Mowla A, Karimi M, Afrasiabi A, De Sanctis V. Prevalence of diabetes mellitus and impaired glucose tolerance in beta-thalassemia patients with and without hepatitis C virus infection.Pediatr Endocrinol Rev. 2004;2 (Suppl 2):282-824.
  66. De Sanctis V, Soliman AT, Elsedfy H, Yaarubi SA, Skordis N, Khater D, El Kholy M, Stoeva I, Fiscina B, Angastiniotis M, Daar S, Kattamis C. The ICET-A Recommendations for the Diagnosis and Management of Disturbances of Glucose Homeostasis in Thalassemia Major Patients Mediterr J Hematol Infect Dis. 2016 Oct 28;8(1):e2016058. https://doi.org/10.4084/mjhid.2016.058 PMid:27872738 PMCid:PMC5111521
  67. Noetzli LJ, Mittelman SD, Watanabe RM, Coates TD, Wood JC. Pancreatic iron and glucose dysregulation in thalassemia major. Am J Hematol 2012;87:155-160. https://doi.org/10.1002/ajh.22223 PMid:22120775
  68. Wankanit S, Chuansumrit A, Poomthavorn P, Khlairit P, Pongratanakul S, Mahachoklertwattana P. Acute Effects of Blood Transfusion on Insulin Sensitivity and Pancreatic β-Cell Function in Children with β-Thalassemia/Hemoglobin E Disease. J Clin Res Pediatr Endocrinol. 2018;10:1-7. https://doi.org/10.4274/jcrpe.4774 PMid:28739553 PMCid:PMC5838366
  69. Petersen DR. Alcohol, iron-associated oxidative stress, and cancer. Alcohol. 2005;35:243-249. https://doi.org/10.1016/j.alcohol.2005.03.013 PMid:16054986
  70. Hu W, Feng Z, Eveleigh J, Iyer G, Pan J, Amin S, Chung FL, Tang MS. The major lipid peroxidation product, trans-4-hydroxy-2-nonenal, preferentially forms DNA adducts at codon 249 of human p53 gene, a unique mutational hotspot in hepatocellular carcinoma. Carcinogenesis. 2002;23:1781-1789. https://doi.org/10.1093/carcin/23.11.1781 PMid:12419825
  71. Barbieri SS, Zacchi E, Amadio P, Gianellini S, Mussoni L, Weksler BB, Tremoli E. Cytokines present in smokers' serum interact with smoke components to enhance endothelial dysfunction. Cardiovasc Res. 2011;90:475-483. https://doi.org/10.1093/cvr/cvr032 PMid:21285293
  72. Díez Piña JM, Fernández Aceñero MJ, Llorente Alonso MJ, Díaz Lobato S, Mayoralas Alises S, Pérez Rodríguez E, Alvaro Álvarez D, Flórez Horcajada A, Pérez Rojo R. Tumor necrosis factor as an early marker of inflammation in healthy smokers. Med Clin (Barc). 2012;139:47-53. https://doi.org/10.1016/j.medcli.2011.11.032 PMid:22401725
  73. Valenca SS, Silva Bezerra F, Lopes AA, Romana-Souza B, Marinho Cavalcante MC, Lima AB, Gonçalves Koatz VL, Porto LC. Oxidative stress in mouse plasma and lungs induced by cigarette smoke and lipopolysaccharide. Environ Res. 2008;108:199-204. https://doi.org/10.1016/j.envres.2008.07.001 PMid:18721919
  74. Purohit V, Rapaka R, Kwon OS, Song BJ. Roles of alcohol and tobacco exposure in the development of hepatocellular carcinoma. Life Sci. 2013;92:3-9. https://doi.org/10.1016/j.lfs.2012.10.009 PMid:23123447 PMCid:PMC3822918
  75. Conti F, Buonfiglioli F, Scuteri A , Crespi C, Bolondi L, Caraceni P, Foschi FG, Lenzi M, Mazzella G, Verucchi G, Andreone P, Brillanti S. Early occurrence and recurrence of hepatocellular carcinoma in HCV related cirrhosis treated with direct-acting antivirals. J Hepatol. 2016;65:727-733. https://doi.org/10.1016/j.jhep.2016.06.015 PMid:27349488
  76. Kozbial K, Moser S, Schwarzer R, Laferl H, Al-Zoairy R, Stauber R, Stättermayer AF, Beinhardt S, Graziadei I, Freissmuth C, Maieron A, Gschwantler M, Strasser M, Peck-Radosalvjevic M, Trauner M, Hofer H, Ferenci P. Unexpected high incidence of hepatocellular carcinoma in cirrhotic patients with sustained virologic response following interferon-free direct-acting antiviral treatment. J Hepatol. 2016; 65:856-858. https://doi.org/10.1016/j.jhep.2016.06.009 PMid:27318327
  77. Cardoso H, Vale AM, Rodrigues S, Gonçalves R, Albuquerque A, Pereira P, Lopes S, Silva M, Andrade P, Morais R, Coelho R, Macedo G. High incidence of hepatocellular carcinoma following successful interferonfree antiviral therapy for hepatitis C associated cirrhosis. J Hepatol. 2016; 65:1070-1071. https://doi.org/10.1016/j.jhep.2016.07.027 PMid:27476768
  78. Reig M, Mariño Z, Perello C, Iñarrairaegui M, Ribeiro A, Lens S, Díaz A, Vilana R, Darnell A, Varela M, Sangro B, Calleja JL, Forns X, Bruix J.Unexpected high rate of early tumor recurrence in patients with HCV-related HCC undergoing interferon-free therapy. J Hepatol. 2016;65:719-726.  https://doi.org/10.1016/j.jhep.2016.04.008 PMid:27084592
  79. ANRS collaborative study group on hepatocellular carcinoma (ANRS CO22 HEPATHER, CO12 CirVir and CO23 CUPILT cohorts). Lack of evidence of an effect of direct-acting antivirals on the recurrence of hepatocellular carcinoma: Data from three ANRS cohorts. J Hepatol.2016;65:734-740. https://doi.org/10.1016/j.jhep.2016.05.045 PMid:27288051
  80. Adhoute X, Castellani P, Bourlière M. Impact of direct-acting antiviral agents on the risk for hepatocellular carcinoma. Transl Gastroenterol Hepatol 2017;2:110. https://doi.org/10.21037/tgh.2017.12.04 PMid:29354767 PMCid:PMC5762991
  81. Soliman A, De Sanctis V, Yassin M. Vitamin d status in thalassemia major: an update. Mediterr J Hematol Infect Dis. 2013 Sep 2;5(1):e2013057.  https://doi.org/10.4084/mjhid.2013.057 PMid:24106607 PMCid:PMC3787712
  82. Wu DB, Wang ML, Chen EQ, Tang H. New insights into the role of vitamin D in hepatocellular carcinoma. Expert Rev Gastroenterol Hepatol. 2018;12:287-294. https://doi.org/10.1080/17474124.2018.1406307 PMid:29140126
  83. Chiang KC, Yeh CN, Chen MF, Chen TC. Hepatocellular carcinoma and vitamin D: a review. J Gastroenterol Hepatol. 2011;26:1597-603. https://doi.org/10.1111/j.1440-1746.2011.06892.x PMid:21880026
  84. Omar A, Abou-Alfa GK, Khairy A, Omar H. Risk factors for developing hepatocellular carcinoma in Egypt. Chin Clin Oncol. 2013 Dec;2(4):43. https://doi.org/10.3978/j.issn.2304-3865.2013.11.07.
  85. Tsuchiya N, Sawada Y, Endo I, Saito K, Uemura Y, NakatsuraT. Biomarkers for the Early Diagnosis of Hepatocellular Carcinoma. World J Gastroenterol. 2015; 21:10573-10583. https://doi.org/10.3748/wjg.v21.i37.10573 PMid:26457017 PMCid:PMC4588079
  86. Makiyama A, Itoh Y, Kasahara A, Imai Y, Kawata S, Yoshioka K, Tsubouchi H, Kiyosawa K, Kakumu S, Okita K, Hayashi N, Okanoue T. Characteristics of patients with chronic hepatitis C who develop hepatocellular carcinoma after a sustained response to interferon therapy. Cancer. 2004;101:1616-1622. https://doi.org/10.1002/cncr.20537 PMid:15378504
  87. Ryder SD; British Society of Gastroenterology. Guidelines for the diagnosis and treatment of hepatocellular carcinoma (HCC) in adults. Gut. 2003; 52 (Suppl 3):1-8. https://doi.org/10.1136/gut.52.suppl_3.iii1 PMid:12692148 PMCid:PMC1867754
  88. Sanyal AJ, Yoon SK, Lencioni R. The etiology of hepatocellular carcinoma and consequences for treatment. Oncologist. 2010;15 (Suppl 4):14-22. https://doi.org/10.1634/theoncologist.2010-S4-14 PMid:21115577
  89. Singal A, Volk ML, Waljee A, Salgia R, Higgins P, Rogers MA, Marrero JA.Meta-analysis: surveillance with ultrasound for early-stage hepatocellular carcinoma in patients with cirrhosis. Aliment Pharmacol Ther. 2009;30:37-47. https://doi.org/10.1111/j.1365-2036.2009.04014.x PMid:19392863 PMCid:PMC6871653
  90. Sciancalepore D, Zingaro MT, Luglio CV, Sabba C, Napoli N. Hepatocellular Carcinoma: Known and Emerging Risk Factors. J Cancer Ther. 2018;9:417-437. https://doi.org/10.4236/jct.2018.95037
  91. Lencioni R, Piscaglia F, Bolondi L. Contrast-enhanced ultrasound in the diagnosis of hepatocellular carcinoma. J Hepatol. 2008;48:848-857. https://doi.org/10.1016/j.jhep.2008.02.005 PMid:18328590
  92. Jo PC, Jang HJ, Burns PN, Burak KW, Kim TK, Wilson SR. Integration of contrast-enhanced US into a multimodality approach to imaging of nodules in a cirrhotic liver: how i do it. Radiology 2017;282:317- 331. https://doi.org/10.1148/radiol.2016151732 PMid:28099108
  93. Nakayama Y, Inoue T, Sakamoto M, Enomoto N. Liver stiffness measurement for risk assessment of hepatocellular carcinoma. Hepatol Res. 2015;45:523-532. https://doi.org/10.1111/hepr.12377 PMid:24961848
  94. Marrero JA, Kulik LM, Sirlin CB, Zhu AX, Finn RS, Abecassis MM, Roberts LR, Heimbach JK. Diagnosis, Staging, and Management of Hepatocellular Carcinoma: 2018 Practice Guidance by the American Association for the Study of Liver Diseases. Hepatology. 2018; 68:723-750. https://doi.org/10.1002/hep.29913 PMid:29624699
  95. Santi V, Trevisani F, Gramenzi A, Grignaschi A, Mirici-Cappa F, Del Poggio P, Di Nolfo MA, Benvegnù L, Farinati F, Zoli M, Giannini EG, Borzio F, Caturelli E, Chiaramonte M, Bernardi M; Italian Liver Cancer (ITA.LI.CA) Group. Semiannual surveillance is superior to annual surveillance for the detection of early hepatocellular carcinoma and patient survival. J Hepatol. 2010; 53:291-297. https://doi.org/10.1016/j.jhep.2010.03.010 PMid:20483497
  96. Simmonds P, Bukh J, Combet C, Deléage G, Enomoto N, Feinstone S, Halfon P, Inchauspé G, Kuiken C, Maertens G, Mizokami M, Murphy DG, Okamoto H, Pawlotsky JM, Penin F, Sablon E, Shin-I T, Stuyver LJ, Thiel HJ, Viazov S, Weiner AJ, Widell A. Consensus proposals for a unified system of nomenclature of hepatitis C virus genotypes. Hepatology.2005;42:962-973. https://doi.org/10.1002/hep.20819 PMid:16149085
  97. Mondelli MU, Silini E. Clinical significance of hepatitis C virus genotypes. J Hepatol. 1999;31 (Suppl 1):65-70. https://doi.org/10.1016/S0168-8278(99)80377-8
  98. Yang JD, Roberts LR. Hepatocellular carcinoma: a global view. Nat Rev Gastroenterol Hepatol. 2010;7:448-458. https://doi.org/10.1038/nrgastro.2010.100 PMid:20628345 PMCid:PMC3926946
  99. Bedossa P. Intraobserver and interobserver variations in liver biopsy interpretation in patients with chronic hepatitis C. Hepatology. 1994; 20:15-20. https://doi.org/10.1002/hep.1840200104
  100. Bedossa P, Dargere D, Paradis V. Sampling variability of liver fibrosis in chronic hepatitis C. Hepatology. 2003; 38:1449-1457. https://doi.org/10.1053/jhep.2003.09022 PMid:14647056
  101. Mikolasevic I, Orlic L, Franjic N, Hauser G, Stimac D, Milic S. Transient elastography [FibroScan ®] with controlled attenuation parameter in the assessment of liver steatosis and fibrosis in patients with nonalcoholic fatty liver disease - Where do we stand? World J Gastroenterol. 2016;22:7236-7251. https://doi.org/10.3748/wjg.v22.i32.7236 PMid:27621571 PMCid:PMC4997649
  102. Bedossa P, Poynard T. An algorithm for the grading of activity in chronic hepatitis C. The METAVIR Cooperative Study Group. Hepatology. 1996; 24, 289- 293. https://doi.org/10.1002/hep.510240201 PMid:8690394
  103. Castera L. Noninvasive methods to assess liver disease in patients with hepatitis B or C. Gastroenterology. 2012; 142:1293-1302. https://doi.org/10.1053/j.gastro.2012.02.017 PMid:22537436

[TOP]