Bone mineral density in adult thalassaemias: a retrospective longitudinal study.

OBJECTIVES: In this study, we aim to evaluate the long-term impact of thalassaemia on bone mineral density (BMD) through sequential analysis, compare changes in BMD values between male and female patients and find any correlation between BMD and biochemical markers in the adult thalassaemia group. BMD is a bone mineral density test using dual-energy X-ray to measure calcium hydroxyapatite per unit of bone, reflecting bone strength. METHODS: We conducted a longitudinal retrospective observational cohort study to determine the changes in BMD values and biochemical parameters in adult thalassaemia patients. BMD was assessed at the lumbar spine (L1-L4) and proximal femora using Hologic's bone dual-energy X-ray absorptiometry. Five serial BMD values were retrieved from electronic records. Biochemical parameters, including serum calcium, phosphorus and 25-hydroxyvitamin D levels, were also assessed. RESULTS: A total of 108 patients (47 males and 61 females; median age: 44 years) with thalassaemia major 71 patients, intermedia 20 patients, haemoglobin E disease 14 patients and thalassaemia-alpha three patients were included. The incidence of low BMD in patients with thalassaemia increased from 64 to 74% over three decades of analysis. Females and thalassaemia major patients had lower hip BMD values and corresponding Z -scores. CONCLUSION: There is a progressive decline in BMD values in adult thalassaemia, which was apparent in female thalassaemia major patients. No changes in biochemical parameters, however, were observed over long-term assessments.

Application of the magnetic resonance 3D multiecho Dixon sequence for quantifying hepatic iron overload and steatosis in patients with thalassemia.

OBJECTIVE: To investigate the feasibility and diagnostic efficacy of a 3D multiecho Dixon (qDixon) research application for simultaneously quantifying the liver iron concentration (LIC) and steatosis in thalassemia patients. MATERIALS AND METHODS: This prospective study enrolled participants with thalassemia who underwent 3 T MRI of the liver for the evaluation of hepatic iron overload. The imaging protocol including qDixon and conventional T2* mapping based on 2D multiecho gradient echo (ME GRE) sequences respectively. Regions of interest (ROIs) were drawn in the liver on the qDixon maps to obtain R2* and proton density fat fraction (PDFF). The reference R2* value was measured and calculated on conventional T2* mapping using the CMRtools software. Correlation analysis, Linear regression analysis, and Bland-Altman analysis were performed. RESULTS: 84 patients were finally included in this study. The median R2*-ME-GRE was 366.97 (1/s), range [206.68 (1/s), 522.20 (1/s)]. 8 patients had normal hepatic iron deposition, 16 had Insignificant, 42 had mild, 18 had moderate. The median of R2*-qDixon was 376.88 (1/s) [219.33 (1/s), 491.75 (1/s)]. A strong correlation was found between the liver R2*-qDixon and the R2*-ME-GRE (r = 0.959, P < 0.001). The median value of PDFF was 1.76% (1.10%, 2.95%). 8 patients had mild fatty liver, and 1 had severe fatty liver. CONCLUSION: MR qDixon research sequence can rapidly and accurately quantify liver iron overload, that highly consistent with the measured via conventional GRE sequence, and it can also simultaneously detect hepatic steatosis, this has great potential for clinical evaluation of thalassemia patients.

Modified Magnetic Resonance Imaging Burden of Cerebral Small Vessel Disease and Related Risk Factors in Patients With Thalassemia.

OBJECTIVE: This study aimed to explore the burden of magnetic resonance imaging (MRI) of cerebral small vessel disease (CSVD) in patients with thalassemia and related risk factors. METHODS: The clinical data and MRI of patients with thalassemia were retrospectively analyzed, and non-thalassemia controls with matched sex and age were selected. The modified MRI burden of CSVD included recent small subcortical infarct, presumed vasogenic white matter hyperintensity, presumed vasogenic lacunae, perivascular space (PVS), and brain atrophy. RESULTS: This study included 110 patients in each of the thalassemia and control groups. There was no significant difference in sex, age, and common cerebrovascular disease risk factors between the 2 groups. The patients with thalassemia had a higher red blood cell count and lower content of hemoglobin. The PVS and modified MRI burden scores in the thalassemia group were higher than in the control group. With the increase in age, patients with thalassemia have a more severe CSVD burden. CONCLUSION: Patients with thalassemia have a heavier modified MRI burden of CSVD than non-thalassemia patients, particularly PVS, and aging is an important risk factor for CSVD changes.

CHMMOTv1 - cardiac and hepatic multi-echo (T2*) MRI images and clinical dataset for Iron overload on thalassemia patients.

INTRODUCTION: Regarding deep learning networks in medical sciences for improving diagnosis and treatment purposes and the existence of minimal resources for them, we decided to provide a set of magnetic resonance images of the cardiac and hepatic organs. DATABASE DESCRIPTION: The dataset included 124 patients (67 women and 57 men) with thalassemia (THM), the age range of (5-52) years. Patients were divided into two groups: with follow-up (1-5 times) at time intervals of about (5-6) months and without follow-up. T2* and, R2* values, the results of the Cardiac and Hepatic overload report (normal, mild, moderate, severe), and laboratory tests including Ferritin, Bilirubin (D, and T), AST, ALT, and ALP levels were provided as an Excel file. Also, the details of the patients' Echocardiogram data have been made available. This dataset CHMMOTv1) has been published in Mendeley Dataverse and also is accessible through the web at: http://databiox.com .

Magnetic resonance imaging assessment of the changes of cardiac and hepatic iron load in thalassemia patients before and after hematopoietic stem cell transplantation.

To investigate the value of T2* technique on 3.0 T magnetic resonance imaging (MRI) in evaluating the changes of cardiac and hepatic iron load before and after hematopoietic stem cell transplantation (HSCT) in patients with thalassemia (TM), the 141 TM patients were divided into 6 group for subgroup analysis: 6, 12, 18, 24 and > 24 months group, according to the postoperative interval. The T2* values of heart and liver (H-T2*, L-T2*) were quantified in TM patients before and after HSCT using 3.0 T MRI T2* technology, and the corresponding serum ferritin (SF) was collected at the same time, and the changes of the three before and after HSCT were compared. The overall H-T2* (P = 0.001) and L-T2* (P = 0.041) of patients after HSCT were higher than those before HSCT (mean relative changes = 19.63%, 7.19%). The H-T2* (P < 0.001) and L-T2* (P < 0.001) > 24 months after HSCT were significantly higher than those before HSCT (mean relative changes = 69.19%, 93.73%). The SF of 6 months (P < 0.001), 12 months (P = 0.008), 18 months (P = 0.002) and > 24 months (P = 0.001) were significantly higher than those before HSCT (mean relative changes = 57.93%, 73.84%, 128.51%, 85.47%). There was no significant improvement in cardiac and liver iron content in TM patients within 24 months after HSCT, while the reduction of cardiac and liver iron content in patients is obvious when > 24 months after HSCT.

Left Ventricular Myocardial Dysfunction Evaluation in Thalassemia Patients Using Echocardiographic Radiomic Features and Machine Learning Algorithms.

Heart failure caused by iron deposits in the myocardium is the primary cause of mortality in beta-thalassemia major patients. Cardiac magnetic resonance imaging (CMRI) T2* is the primary screening technique used to detect myocardial iron overload, but inherently bears some limitations. In this study, we aimed to differentiate beta-thalassemia major patients with myocardial iron overload from those without myocardial iron overload (detected by T2*CMRI) based on radiomic features extracted from echocardiography images and machine learning (ML) in patients with normal left ventricular ejection fraction (LVEF > 55%) in echocardiography. Out of 91 cases, 44 patients with thalassemia major with normal LVEF (> 55%) and T2* ≤ 20 ms and 47 people with LVEF > 55% and T2* > 20 ms as the control group were included in the study. Radiomic features were extracted for each end-systolic (ES) and end-diastolic (ED) image. Then, three feature selection (FS) methods and six different classifiers were used. The models were evaluated using various metrics, including the area under the ROC curve (AUC), accuracy (ACC), sensitivity (SEN), and specificity (SPE). Maximum relevance-minimum redundancy-eXtreme gradient boosting (MRMR-XGB) (AUC = 0.73, ACC = 0.73, SPE = 0.73, SEN = 0.73), ANOVA-MLP (AUC = 0.69, ACC = 0.69, SPE = 0.56, SEN = 0.83), and recursive feature elimination-K-nearest neighbors (RFE-KNN) (AUC = 0.65, ACC = 0.65, SPE = 0.64, SEN = 0.65) were the best models in ED, ES, and ED&ES datasets. Using radiomic features extracted from echocardiographic images and ML, it is feasible to predict cardiac problems caused by iron overload.

Quantification of cardiac iron in patients with thalassemia with 3-T MRI calibrated by 1.5-T MRI.

BACKGROUND: Due to the small sample size of many studies, it remained unclear what standardized reference range the T2* cutoff at 3 T would be used to assess the severity of cardiac iron load. In addition, the number of patients with moderate to severe cardiac iron load was small in some studies, especially the sample of patients with severe cardiac iron load. PURPOSE: To explore the feasibility, reproducibility, and reliability of using T2* values in quantifying cardiac iron load in patients with thalassemia at 3 T. MATERIAL AND METHODS: A total of 122 patients with thalassemia underwent cardiac T2* imaging at both 1.5 T and 3 T. Cardiac R2* (1000/T2*) values of the 100 patients at 3 T were fitted against the values at 1.5 T using linear regression and the prediction equation was derived. The remaining 22 cases were used to test the prediction accuracy of the equation. RESULTS: The combined R2* values exhibited a strong linear relationship between 1.5 T and 3 T (r = 0.830，P<0.001). At the center, it had a slope of 1.348 and an intercept of 37.279. According to the equation, the truncated T2* values of cardiac iron overload and cardiac heavy iron overload at 3 T were <10 ms and <6 ms, respectively. The two truncated T2* values were used to diagnose different levels of cardiac iron overloaded of 22 patients at 3 T; the accuracy rates were 95.5% and 100.0%, respectively. CONCLUSION: T2* quantification of cardiac iron load at 3 T MRI resulted to be feasible, reproducible, and reliable.

Correlation of Transient Elastography With MRI T2* and Serum Ferritin Levels in Children With Transfusion-Dependent Thalassemia.

OBJECTIVES: We investigated the correlation of transient elastography (TE) with MRI R2* values and serum ferritin in patients with transfusion-dependent thalassemia (TDT). METHODS: We reviewed hospital records of 59 patients with TDT aged ³8 years without any evidence of chronic liver disease and who had fibroscan within 3 months of MRI T2*, who seen at our center between January, 2014 and December, 2019. Spearman correlation and linear regression analysis were used to evaluate the correlation between TE liver stiffness measurements and R2* MRI values and with serum ferritin. RESULTS: Mean (SD) age of the subjects was 13.0 (3.1) years and body mass index was 16.6 (2.3) kg/m2. Mean liver stiffness measurement, MRI T2*(3T), corresponding MRI R2*(3T), and ferritin values were 6.55 (3.10) kPa, 3.4 (4.6) milliseconds, 616.20 (383.9) Hz, and 2874.69 (1570.7) ng/mL, respectively. TE measurements correlated with MRI R2* values (r=0.61; P=0.001) and with serum ferritin level (r=0.59, P=0.001). CONCLUSION: TE is a reliable tool to estimate hepatic iron overload in patients with TDT.

The Importance of Functional and Feature-Tracking Cardiac MRI Parameters in Prediction of Adverse Cardiac Events and Cardiac Mortality in Thalassemia Patients.

RATIONALE AND OBJECTIVES: Despite some investigations about the role of cardiovascular magnetic resonance (CMR) imaging in thalassemia, there are a few studies regarding the feature-tracking (FT). We evaluated the role of T2*, functional, and FT values for the determining of adverse cardiac events (ACE). METHODS: One-hundred-fifty-nine patients with thalassemia-major (49.7% female, mean-age = 32 ± 9.8 year) were followed for 8 - 64 (median = 36) months. CMR derived functional, FT, and T2* as well as ACE (heart failure hospitalization, cardiac mortality, pulmonary hypertension, and arrhythmias) were recorded. Also, variables were analyzed for cardiac death prediction separately. RESULTS: Seventeen patients (10.7%) developed ACE. The right-ventricular ejection fraction (RVEF) was the strongest indicator of ACE (OR: 0.85, 95% - CI: 0.790 - 0.918; p < 0.001) and cardiac mortality (OR: 0.88, 95%-CI: 0.811 - 0.973; p = 0.01). RVEF ≤ 39% and ≤ 37% predicted ACE and mortality with sensitivity of 62.5% and 71.43% and specificity of 95.77% and 93.38%, respectively. Additionally, myocardial-T2* was a predictor of mortality (OR: 0.90, 95%-CI: 0.814 - 0.999; p = 0.04). T2* ≤ 10 months predicted death with 85.71% sensitivity and 85.91% specificity. RV global longitudinal strain (GLS) was the strongest strain parameter for the indication of ACE and death (OR: 0.81, 95%-CI: 0.740 - 0.902; p < 0.001 and OR: 0.81, 95%- CI: 0.719 - 0.933; p = 0.003, respectively). RV GLS ≤ 16.43% and ≤ 15.63% determined ACE and death with sensitivity of 52.94% and 71.43% and specificity of 90%, respectively. CONCLUSION: Our results underscore the role of FT and non-contrast CMR parameters as valuable markers of ACE in thalassemia.

Forager and farmer evolutionary adaptations to malaria evidenced by 7000 years of thalassemia in Southeast Asia.

Thalassemias are inherited blood disorders that are found in high prevalences in the Mediterranean, Southeast Asia and the Pacific. These diseases provide varying levels of resistance to malaria and are proposed to have emerged as an adaptive response to malaria in these regions. The transition to agriculture in the Holocene has been suggested to have influenced the selection for thalassemia in the Mediterranean as land clearance for farming encouraged interaction between Anopheles mosquitos, the vectors for malaria, and human groups. Here we document macroscopic and microscopic skeletal evidence for the presence of thalassemia in both hunter-gatherer (Con Co Ngua) and early agricultural (Man Bac) populations in northern Vietnam. Firstly, our findings demonstrate that thalassemia emerged prior to the transition to agriculture in Mainland Southeast Asia, from at least the early seventh millennium BP, contradicting a long-held assumption that agriculture was the main driver for an increase in malaria in Southeast Asia. Secondly, we describe evidence for significant malarial burden in the region during early agriculture. We argue that the introduction of farming into the region was not the initial driver of the selection for thalassemia, as it may have been in other regions of the world.

Extensive Extramedullary Hematopoiesis in Thalassemia: Soft Tissue Uptake on 99mTc-MDP SPECT/CT.

We report the case of a 45-year-old man with thalassemia major referred for a bone scan as a workup for generalized bone pain. Tc-MDP SPECT CT showed multiple elongated soft tissue masses along the inner aspects of ribs and along both sides of dorsal spine with calcification and mild tracer uptake. There was also a large lobulated presacral soft tissue mass displacing the bladder and involving the adjacent pelvic bone. Biopsy of mildly avid tracer uptake in pelvic soft tissue mass was extramedullary hematopoiesis confirmed by biopsy.

Thalassemia in the emergency department: special considerations for a rare disease.

Thalassemia is characterized by a defect in the synthesis of one or more of the globin subunits of hemoglobin. This defect results in imbalance in the α/ß-globin chain ratio, ineffective erythropoiesis, chronic hemolytic anemia, and iron overload. With advances in diagnosis, treatment, and transfusion support, the prognosis of patients with thalassemia has improved over the past few decades. An increasing number of patients with thalassemia is living with long-term complications, including cardiomyopathy, chronic liver disease, endocrinopathy, and infections. In this paper, we review common complications that bring the patient with thalassemia to urgent or emergent medical attention. We also discuss the aspects of emergency care that are most relevant while caring for the patient with thalassemia in the emergency department.

N-acetylcysteine Restored Heart Rate Variability and Prevented Serious Adverse Events in Transfusion-dependent Thalassemia Patients: a Double-blind Single Center Randomized Controlled Trial.

Regular blood transfusions in transfusion-dependent thalassemia (TDT) patients can lead to iron overload, causing oxidative stress and sympathovagal imbalance, resulting in increased cardiac complications. We hypothesized that administrating of N-acetylcysteine (NAC) prevents serious adverse events including cardiac complications in TDT patients by reducing systemic oxidative stress and balancing cardiac sympathovagal control. This study was double-blind, randomized control trial, investigating in 59 Thai TDT patients. After randomization, the participants were divided into two groups. The control group received standard care of TDT patient plus placebo, whereas the intervention group received 600 mg of NAC orally for six months. Serum 8-isoprostane, TNF-alpha, IL-10, 24-hour ECG monitoring, echocardiograms and the incidence of thalassemia-related complications were collected. At baseline, no significant difference in any parameters between the control and the intervention groups. At the end of intervention, the incidence of serious adverse events (i.e. infection, worsening thalassemia) was significantly higher in the control group when compared with the intervention group (24.1% vs. 3.3%, p=0.019) (Chi-square test; absolute risk reduction=20.8%, number needed to treat=4.8). The control group also had significantly lower time-dependent HRV parameters, compared with the intervention group (p=0.025 and 0.030, independent t-test). Treatment with NAC restored HRV and reduced serious adverse event in TDT patients, however, no difference in cardiac complications could be demonstrated. NAC could prevent serious adverse events in TDT patients. The proposed mechanism might be the balancing of sympathovagal control.

Effect of different iron chelation regimens on bone mass in transfusion-dependent thalassemia patients.

Objectives: Iron overload might lead to bone loss in transfusion-dependent beta-thalassemia (TDT) patients. To investigate the role of iron chelation therapy (ICT) on bone mineral density (BMD) of TDT patients suffering from iron overload, the authors compared the efficacy of five different iron chelation regimens through assessing serum ferritin and BMD.Methods: In 256 consecutive TDT patients, BMD was measured by dual-energy X-ray absorptiometry in lumbar spine and femoral neck regions. Treatment outcome of five iron chelation regimens including Deferoxamine (DFO), Deferiprone (DFP), Deferasirox (DFX), and combination therapy was evaluated to compare the mean differences of serum ferritin and BMD indices pre- and post-treatment during 12-months follow-up period.Results: No significant difference was observed in DXA characteristics and serum ferritin level changes between ICT groups, but combination of DFO and DFX had the best outcome in improving bone mass through assessing each group individually.Conclusion: Combination therapy with DFX and DFO had the highest impact on reducing serum ferritin, however insignificant, and improving bone loss in both lumbar spine and femoral neck in comparison with other regimens. A randomized prospective clinical trial is advised to accurately assess the efficacy of iron chelation regimens on BMD measurements of TDT patients.

Prevalence and prognostic impact of left ventricular non-compaction in patients with thalassemia.

A high incidence of isolated left ventricular non-compaction (LVNC) has been reported in previous studies on smaller cohorts of patients with thalassemia by cardiac MRI but the clinical impact of the finding is unknown. This prospective cohort study evaluates the prevalence and clinical implication of the finding. Prospective cohort study with enrollment of all consecutive cases with thalassemia referred for cardiac MRI from September 2007 to November 2014. The presence of LVNC was assessed according to the Petersen method and the Jacquier method, with the proposed changes by Fazio, Grothoff, and Chiodi. A clinical follow-up was performed in all patients. We included 560 patients with thalassemia (473 with thalassemia major and 87 with thalassemia intermedia: mean age 31.9 ± 10.6 years, male/female = 250/310). A total number of 1683 MRI tests were performed. A diagnosis of LVNC was determined according to adopted MR criteria in 44 patients (7.9%). Patients with LVNC had a significantly lower ejection fraction (52.68 ± 5.17% vs. 56.90 ± 6.34%; p = 0.0005) and greater indexed LV ESV (48.16 ± 10.03 ml/m2 vs. 40.02 ± 10.06 ml/m2; p = 0.0022). After a mean follow-up time was 5.1 years, no significant change of MR parameters was detected as well as no clinical adverse events. LVNC is relatively frequent in patients with thalassemia. However, it is not associated with a worsening of LV function and adverse events after a long-term follow-up.

Comparative effectiveness of alendronate and zoledronic acid on bone mass improvement in transfusion-dependent thalassemia patients.

Thalassemia, as the most prevalent genetic blood disorder, has many associated comorbidities including low bone mass. We studied the comparative effectiveness of alendronate (AL) and zoledronic acid (ZOL) on bone mass improvement in transfusion-dependent thalassemia (TDT) patients a year after treatment. Three hundred seventy-five TDT patients with low bone mass were enrolled in this study. After a year of treatment with either AL or ZOL, a second bone mineral density (BMD) test was ordered to compare the effectiveness of the two aforementioned drugs. Body mass index (BMI), physical activity, sun exposure, and biochemical laboratory data were also considered as associated factors in this study. The BMD test of both groups was almost the same at the baseline and it increased comparably after a year of treatment with AL and ZOL. However, there was a significant difference in lumbar spine BMD delta Z score between both groups of female patients. ZOL was more effective in increasing the lumbar spine BMD of female patients. The choice of bisphosphonates therapy (oral versus parenteral) should be individually selected by considering patient's preference, compliance and the physician's decision. Given the longer administration interval, and TDT patients' compliance issue, it is justified to recommend ZOL as the drug of choice for patients suffering from low bone mass.

Erythropoiesis and Iron Homeostasis in Non-Transfusion-Dependent Thalassemia Patients with Extramedullary Hematopoiesis.

BACKGROUND: Extramedullary hematopoiesis (EMH) is common in non-transfusion-dependent thalassemia (NTDT) patients. Clinical presentations of EMH vary as MRI screening is not feasible. Hence, serum biomarkers are used to predict the risk of EMH. MATERIALS AND METHODS: 52 NTDT patients, including 26 EMH (+) and 26 EMH (-), together with 26 healthy controls, were enrolled in this case-control study from 2013 to 2016. EMH was confirmed by computed tomography or MRI. Demographic, transfusion, genetic, laboratory, and liver iron concentration (LIC) data, as well as clinical complications, were analyzed. RESULTS: EMH (+) patients had significantly higher serum ferritin (SF), growth differentiation factor 15 (GDF15), and erythropoietin (EPO) levels compared with EMH (-) patients and controls. The levels of erythroferrone (ERFE), hepcidin, and sTfR did not differ significantly between EMH (+) and EMH (-) patients (p>0.05). In NTDT patients, serum ERFE was not related to SF, LIC, hepcidin, sTfR, EPO, GDF15, and Hb levels. GDF15, EPO concentrations, and GDF15 to sTfR and GDF15 to EPO ratios are able to determine the presence of EMH with considerable sensitivity and specificity. CONCLUSIONS: GDF15, EPO, and GDF15 to EPO and GDF15 to sTfR ratios are potential biomarkers for the early prediction of NTDT in patients who are at risk for EMH.

Imbalance of erythropoiesis and iron metabolism in patients with thalassemia.

Aim: This study aimed to evaluate the imbalance of erythropoiesis and iron metabolism in patients with thalassemia. Methods: 192 patients with non-transfusion-dependent thalassemia (NTDT), 94 patients with transfusion-dependent thalassemia (TDT) and 101 healthy controls were recruited between June 2013 and December 2016 in the Hematology Department, the First Affiliated Hospital of Guangxi Medical University. The groups were compared in terms of levels of erythropoiesis biomarkers [growth differentiation factor 15 (GDF15), erythropoietin (EPO) and soluble transferrin receptor (sTfR)] and of iron overload biomarkers [serum ferritin (SF), liver iron concentration (LIC) and cardiac T2*] and hepcidin. Results: The levels of GDF15, EPO, sTfR, LIC and SF were significantly higher in patients with thalassemia. The levels of GDF15 and EPO were significantly higher in patients with TDT compared to NTDT. Those with iron overload had higher EPO, GDF15, SF and sTfR levels compared with non-iron overload patients. Hepcidin levels and ratios of hepcidin to erythropoietic activity and to iron biomarker levels were lower in patients with ß-thalassemia intermedia or hemoglobin (Hb) E/ß-thalassemia than in patients with HbH disease. The hepcidin levels were correlated negatively with the levels of EPO, GDF15 and sTfR in patients with NTDT and TDT, but correlated positively with SF and Hb levels only in patients with TDT. Conclusions: Patients with thalassemia showed iron overload, reduced hepcidin levels, and a greater extent of ineffective erythropoiesis. The hepcidin levels were more strongly related to ineffective erythropoiesis compared with iron overload. The imbalance between erythropoiesis and iron metabolism differed across different thalassemia types.

Imaging features of thalassaemia.

This article highlights the range of osseous findings that can be encountered as well as the imaging features of extramedullary haematopoiesis. As iron overload remains a major cause of morbidity and mortality in these disorders, we also discuss the MRI evaluation of hepatic and cardiac hemosiderosis, to aid in the optimization of iron chelation therapy. Future imaging use will be dictated by evolving clinical needs, such as in screening for emerging morbidities, including hepatic fibrosis and hepatocellular carcinoma.