Impairment of IFN-α production capacity in patients with hepatitis C virus and the risk of the development of hepatocellular carcinoma

AIM: To determine the utility of interferon (IFN)-alpha production capacity in patients with hepatitis C virus (HCV) infection for the measurement of immuno-surveillance potential and for the early detection of hepatocellular carcinoma (HCC) by investigating the Sendai virus (HVJ) stimulated IFN-alpha production capacity of patients with HCV infection.METHODS: HVJ stimulated IFN-alpha production was determined in a large number of patients with HCV infection and the development of HCC was monitored for 3 years in patients with liver cirrhosis (LC).RESULTS: IFN-alpha production capacity decreases gradually with the progression of liver disease from chronic hepatitis (CH) to HCC. A significant correlation between the duration of HCV infection and impaired IFN-alpha production capacity was observed. IFN-alpha production in patients who developed HCC within 3 years was significantly lower than that of patients who remained in LC without developing HCC.CONCLUSION: Measurement of IFN-alpha production in LC patients may be useful for the early detection of HCC.     

Value of α-fetoprotein in association with clinicopathological features of hepatocellular carcinoma

AIM:To explore the relationship between α-fetoprotein(AFP) and various clinicopathological variables and different staging system of hepatocellular carcinoma(HCC) thoroughly.METHODS:A retrospective cohort study of consecutive patients diagnosed with HCC between January 2008 and December 2009 in West China Hospital was enrolled in our study.The association of serum AFP values with the HCC clinicopathological features was analysed by univariate and multivariate analysis,such as status of hepatitis B virus(HBV) infection,tumor size,tumor number,vascular invasion and degree of tumor differentiation.Also,patients were divided into four groups at the time of enrollment according to different cutoff values for serum value of AFP(≤ 20 μg/L,21-400 μg/L,401-800 μg/L,and ≥ 801 μg/L),to compare the positive rate of patient among four groups stratified by various clinicopathological variables.And the correlation of different kinds of tumor staging systems,such as TNM,Barcelona Clinic Liver Cancer(BCLC) staging classification and China staging,were compared with the serum concentration of AFP.RESULTS:A total of 2304 HCC patients were enrolled in this study totally;the mean serum level of AFP was 555.3 ± 546.6 μg/L.AFP levels were within the normal range(< 20 μg/L) in 27.4%(n = 631) of all the cases.81.4%(n = 1875) patients were infected with HBV,and those patients had much higher serum AFP level compared with non-HBV infection ones(573.9 ± 547.7 μg/L vs 398.4 ± 522.3 μg/L,P < 0.001).The AFP level in tumors ≥ 10 cm(808.4 ± 529.2 μg/L) was significantly higher(P < 0.001) than those with tumor size 5-10 cm(499.5 ± 536.4 μg/L) and with tumor size ≤ 5 cm(444.9 ± 514.2 μg/L).AFP levels increased significantly in patients with vascular invasion(694.1 ± 546.9 μg/L vs 502.1 ± 543.1 μg/L,P < 0.001).Patients with low tumor cell differentiation(559.2 ± 545.7 μg/L) had the significantly(P = 0.007) highest AFP level compared with high differentiation(207.3 ± 420.8 μg/L) and intermediate differe     

Importance of SALL4 in the development and prognosis of hepatocellular carcinoma

AIM: To detect the expression of sal-like protein 4(SALL4) and to explore its relationship with clinicopathological characteristics and prognosis of hepatocellular carcinoma(HCC).METHODS: One hundred and twenty-six samples of HCC tissue, 44 of adjacent noncancerous cirrhotic tissue and 10 of liver hemangioma tissue, were obtained from patients who underwent hepatectomy for HCC at the Fourth Hospital of Hebei Medical University. None of the patients had received any form of treatment before the operation. After resection, all the tissues were fixed in 10% neutral formaldehyde and embedded in paraffin. Expression of SALL4 was detected by immunohistochemistry. Patients were followed up for postoperative survival until February 2014. The relationships between SALL4 expression level and clinicopathological data and prognosis of HCC were analyzed. RESULTS: SALL4 expression was negative in the 10 samples of tissue from liver hemangioma, was weakly positive in the two samples from adjacent noncancerous cirrhotic tissue, and positive in 58 samples of HCC tissues. The differences were statistically significant(P 0.05). Expression of SALL4 was higher in patients with higher α-fetoprotein(AFP) levels, portal vein tumor thrombus, and later clinical stage based on the Barcelona Clinic Liver Cancer classification(P 0.05). Among patients with negative expression, weakly positive expression, positive expression, and strongly positive expression of SALL4, the median survival time was 39, 25, 23, and 9 mo, respectively(P 0.001). When both AFP and SALL4 were detected, patients who were negative for both AFP and SALL4, SALL4-positive only, AFP-positive only, and positive for both AFP and SALL4, had a median survival time of 41, 38, 31, and 12 mo, respectively(P 0.001).CONCLUSION: Expression of SALL4 is relevant to the prognosis of HCC patients. Patients with higher expression levels of SALL4 and AFP have worse prognosis.     

Aerobic exercise improves insulin resistance and decreases body fat and serum levels of leptin in patients with hepatitis C virus

Aim: Hepatitis C virus infection often complicates glucose intolerance, which can be caused by insulin resistance. Aerobic exercise can improve insulin resistance and decrease body fat in patients with diabetes. The aim of the present study is to clarify whether aerobic exercise improves insulin resistance and decreases body fat in patients with chronic hepatitis C (CH‐C). Methods: Seventeen patients with CH‐C received nutrition education at entry and every two months thereafter. The following were evaluated before and after 6 months of walking at least 8000 steps/day monitored using a pedometer that started 2 months after entry: body composition, fat and muscle weight, visceral and subcutaneous fat areas (VFA and SFA, respectively), liver function tests, the Homeostatic Model of Assessment of Insulin Resistance (HOMA‐IR), serum tumor necrosis factor‐alpha (TNF‐α), interleukin (IL)‐6, adiponectin, leptin and the Short Form‐36. Results: Fifteen of the 17 patients completed the study protocol. Bodyweight, body mass index, fat weight, VFA, SFA, alanine aminotransferase level and HOMA‐IR were significantly decreased at the end of the study (P = 0.004, =0.004, =0.008, =0.041, =0.001, =0.023 and =0.002, respectively). Serum levels of TNF‐α, IL‐6 and adiponectin did not change, whereas those of leptin significantly decreased (P = 0.002). Conclusion: Patients with CH‐C could safely walk as aerobic exercise. Furthermore, walking improved insulin resistance and decreased body fat while lowering serum levels of leptin.     

肝癌组织HNF4α mRNA定量检测方法的建立与初步应用

目的 建立一种定量检测肝细胞癌（HCC）组织肝细胞核因子4α（HNF4α）mRNA的方法。方法 取手术获得的HCC组织16例,提取总RNA,逆转录PCR扩增获得全长HNF4α mRNA对应cDNA产物,经TA克隆后,测序确认。根据获得序列,设计检测HNF4α mRNA 引物及MGB荧光探针。以体外转录HNF4α mRNA为标准品,建立一步法 逆转录荧光定量PCR检测方法,同时设定β-actin mRNA为内参对照,最终计算得到组织HNF4α mRNA水平。采用免疫组化染色检测HNF4α蛋白表达,比较HNF4α mRNA水平与HNF4α蛋白表达的对应结果。结果 HCC组织HNF4α mRNA定量标准曲线斜率为-3.237,相关系数r值达0.994,β-actin mRNA定量标准曲线斜率为-3.037,相关系数r值为0.996,表明建立的方法可用于检测HNF4α mRNA定量（V-4α值）,16例HCC组织HNF4α mRNA与HNF4α 蛋白表达结果呈一致性对应关系。结论 我们初步成功建立了定量检测HCC组织HNF4α mRNA水平方法,其意义还需要进一步研究。     

Synergistic association of the copper/zinc ratio under inflammatory conditions with diabetic kidney disease in patients with type 2 diabetes: The Asahi Diabetes Complications Study

Aims/IntroductionWe aimed to study the relationships among the copper (Cu)/zinc (Zn) ratio, inflammatory biomarkers, and the prevalence of diabetic kidney disease (DKD) in patients with type 2 diabetes.Materials and MethodsA cross‐sectional study was performed on 651 patients with type 2 diabetes. DKD was defined as a urinary albumin‐to‐creatinine ratio of ≥30 mg/g creatinine and/or an estimated glomerular filtration rate using cystatin C of < 60 mL/min/1.73 m². Areas under the curves (AUCs), cutoff values, and thresholds for detecting DKD were determined for the Cu/Zn ratio, soluble tumor necrosis factor‐α receptor 1 (sTNFαR1), and high‐sensitivity C‐reactive protein (hsCRP). Patients were categorized by each cutoff value of sTNFαR1 and the Cu/Zn ratio. Odds ratios (ORs) and biological interactions for the prevalence of DKD were determined.ResultsDKD was identified in 220 patients. AUC/optimal cutoff values were 0.777/1300 pg/mL for sTNFαR1, 0.603/1.1648 for the Cu/Zn ratio, and 0.582/305 ng/mL for hsCRP. The ORs for DKD were higher, but not significantly, in the sTNFαR1 < 1300 and Cu/Zn ≥ 1.1648 group, significantly higher in the sTNFαR1 ≥ 1300 and Cu/Zn < 1.1648 group (P < 0.0001), and further synergistically elevated in the sTNFαR1 ≥ 1300 and Cu/Zn ≥ 1.1648 group (P < 0.0001) compared with the sTNFαR1 < 1300 and Cu/Zn < 1.1648 group after multivariable adjustment. Levels of sTNFαR1 were significantly higher in the sTNFαR1 ≥ 1300 and Cu/Zn ≥ 1.1648 group than in the sTNFαR1 ≥ 1300 and Cu/Zn < 1.1648 group (P = 0.0006).ConclusionsUnder an inflammatory initiation signal of elevated serum sTNFαR1 levels, an increase in the Cu/Zn ratio may further exacerbate inflammation and is synergistically associated with a high prevalence of DKD in patients with type 2 diabetes.     

Short- and long-term outcome of interferon therapy for chronic hepatitis B infection

Hepatitis B virus (HBV) infection is a serious clinical problem worldwide. Conventional interferon (IFN)-α has been approved for the treatment of chronic hepatitis B (CHB). Short-term studies have demonstrated that IFN-based therapy is moderately effective in inducing the loss of hepatitis e antigen (HBeAg) or seroconversion (30%-40%) in HBeAg-positive patients and also produces sustained HBV DNA suppression (20%-30%) in HBeAg-negative patients. Many studies have reported a correlation between the HBV genotype and response to IFN treatment. The highest response rate to IFN treatment was found in patients infected with HBV genotype A, followed by HBV genotypes B, C, and D. The long-term effect of IFN-α on CHB has not yet been elucidated. The ability of IFN-α treatment to prevent new cirrhosis, complications associated with cirrhosis, and development of hepatocellular carcinoma (HCC) is controversial. The beneficial effect of IFN-α treatment in reducing the development of HCC has mainly been observed in treatment responders who already have cirrhosis. These inconsistent findings may be attributed to the inevitable limitations of comparisons across studies, including differences in the baseline characteristics of the study and the moderate suppression of HBV replication by IFN-α relative to nucleoside/nucleos(t)ide analogs.     

Immunohistochemical evidence of insulin-like growth factor II in human small hepatocellular carcinoma with hepatitis C virus infection: Relationship to fatty change in carcinoma cells

It has recently been reported that insulin-like growth factor II (IGF-II) may play a role in the pathogenesis of hepatocellular carcinoma (HCC). We studied the relationship between the expression of IGF-II and fatty change in human small HCC using immunohistochemical staining techniques. Liver biopsy specimens were obtained from 35 patients with HCC (consisting of 15 patients with fatty change and 20 patients without fatty change). All patients had serum markers for the hepatitis C virus (HCV) and histological findings obtained from non-tumourous lesions showed liver cirrhosis or chronic active hepatitis. Immunohistochemical staining was performed using a monoclonal antibody against rat IGF-II. A positive immunoreaction was found in 69% (24/35) of HCC. Insulin-like growth factor II was immunodetected in 80% (12/15) of HCC with fatty change but only in 60% (12/20) of those without fatty change. In most cases, IGF-II was not found in hepatocytes from non-tumourous lesions. We believe this to be the first time that IGF-II has been detected immunohistochemically in small HCC derived from HCV infection. This growth factor was more frequently immunodetected in HCC with fatty change than without. As insulin is an essential factor for the metabolism of fatty acids, IGF-II may play an important role in both fatty degeneration and in the proliferation of HCC cells. Furthermore, immunohistochemical IGF-II staining may contribute to the diagnosis of HCC, particularly in early stages accompanied by fatty change.     

Serum α-fetoprotein level at treatment completion is a useful predictor of hepatocellular carcinoma occurrence more than one year after hepatitis C virus eradication by direct-acting antiviral treatment

Direct-acting antivirals (DAAs) have recently been developed to treat hepatitis C virus (HCV) infection, and interferon-free DAA treatment has improved liver function of HCV patients. The risk of hepatocellular carcinoma (HCC) occurrence following HCV eradication has been previously reported, but HCC may have been missed following imaging diagnosis before DAA administration in previous studies. Therefore, the present study aimed to identify definite predictors of HCC occurrence ≥1 year after DAA treatment. Among 956 patients receiving DAAs for HCV infection, 567 patients who achieved sustained virologic response with no history of HCC treatment were enrolled in this study between September 2014 and July 2021. The incidence of HCC in HCV-infected patients ≥1 year following DAA treatment, and the predictors contributing to HCC occurrence were identified using clinical characteristics and blood test results. In the present study, 25 patients developed HCC. The incidence of HCC was 1.4%, 3.2%, 4.9% and 6.8% at 2, 3, 4 and 5 years, respectively, from the end of treatment with DAAs. Multivariate logistic analysis revealed serum α-fetoprotein level at end of treatment (EOT-AFP) >3.8 ng/ml ≥1 year following treatment with DAAs (HR, 9.7; p < .0001) as an independent factor that may contribute to HCC occurrence following DAA treatment. In conclusion, serum EOT-AFP level may serve an important role in determining the risk of HCC occurrence ≥1 year after DAA treatment. Regular examinations are required even if serum EOT-AFP level is low at treatment completion.     

Mucosal healing effect of nilotinib in indomethacin-induced enterocolitis: A rat model

AIM: To investigate the effects of nilotinib in a rat model of indomethacin-induced enterocolitis.METHODS: Twenty-one Wistar albino female rats obtained from Dokuz Eylul University Department of Laboratory Animal Science were divided into the following three groups: control(n = 7), indomethacin(n = 7) and nilotinib(n = 7). A volume of 0.25 m L of physiological serum placebo was administered to the control and indomethacin groups through an orogastric tube for 13 d. To induce enterocolitis, the indomethacin and nilotinib groups received 7.5 m L/kg indomethacin dissolved in 5% sodium bicarbonate and administered subcutaneously in a volume of 0.5 m L twice daily for three days. Nilotinib was administered 20 mg/kg/d in two divided doses to the nilotinib group of rats for 13 d through an orogastric tube, beginning on the same day as indomethacin administration. For 13 d, the rats werefed a standard diet, and their weights were monitored daily. After the rats were sacrificed, the intestinal and colonic tissue samples were examined. The macroscopic and microscopic pathology scores were evaluated. The pathologist stained all tissue samples using terminal deoxynucleotidyl transferase-mediated d UTP-biotin nick-end labeling method. Mucosal crypts and apoptotic cells were quantified. The plateletderived growth factor receptor(PDGFR) α and β scores assessed by immunohistochemical staining method and tissue and serum tumor necrosis factor(TNF) α levels were determined by enzyme-linked immunosorbent assay. RESULTS: Between days 1 and 13, the rats in the nilotinib and indomethacin groups lost significantly more weight than the controls(-11 g vs +14.14 g, P = 0.013;-30 g vs +14.14 g, P = 0.003). In the small intestinal and colonic tissues, the macroscopic scores were significantly lower in the nilotinib group than in the indomethacin group(1.14 ± 0.38 and 7.29 ± 2.98, P = 0.005; 1.14 ± 0.38 and 7.43 ± 2.64, P = 0.001, respectively), but the values of the nilotinib and indomethacin groups were similar to the control group. In the small intestinal and colonic tissues, the microscopic scores were significantly lower in the nilotinib group than in the indomethacin group(3.43 ± 2.99 and 7.67 ± 3.67, P = 0.043; 2.29 ± 0.76 and 8.80 ± 2.68, P = 0.003, respectively), but the values were similar to the control group. The PDGFR β scores in the small intestine and colon were significantly lower in the nilotinib group than in the indomethacin group(1.43 ± 0.79 and 2.43 ± 0.54, P = 0.021; 1.57 ± 0.54 and 3 ± 0, P =0.001), and the values were similar to controls. The colonic PDGFR α scores were significantly lower in the nilotinib group than in the indomethacin group(1.71 ± 0.49 and 3 ± 0, P = 0.001). The colonic apoptosis scores were significantly lower in the controls than in the nilotinib group(1.57 ± 1.13 and 4 ± 1.29, P = 0.007). Furthermore, the serum and tissue TNF-α levels were similar between the nilotinib and indomethacin groups.C O N C L U S I O N : In the indomethacin-induced enterocolitis rat model, nilotinib has a positive effect on the macroscopic and microscopic pathologic scores, ensuring considerable mucosal healing. Nilotinib decreases PDGFR α and β levels and increases the colonic apoptotic scores, but it has no significant effects on weight loss and the TNF-α levels.     

Surveillance of hepatocellular carcinoma in patients with hepatitis C virus infection may improve patient survival.

BACKGROUND: The benefit of surveillance of hepatocellular carcinoma (HCC) for patients with hepatitis C virus (HCV) infection, in terms of long-term survival, has not yet been established. METHODS: A total of 384 consecutive anti-HCV-positive HCC patients admitted to our hospital between January 1991 and October 2003 were enrolled. Patients were categorized into two groups, a surveillance group (182 patients) and a non-surveillance group (202 patients), according to tumor detection in a surveillance program based on periodical examination via ultrasound sonography and alpha fetoprotein determination at 6-month intervals, and their survival rates were compared. RESULTS: Although there were no significant differences in age and Child-Pugh classes between the two groups, the surveillance group exhibited a smaller tumor size (19 vs. 35 mm) and a higher incidence of single HCC (67% vs. 46%), compared with the non-surveillance group (each, P < 0.001). The cumulative survival rate in the surveillance group was higher than that in the non-surveillance group (5 years survival, 46% vs. 32%, P < 0.001). When the survival after correction of the lead-time bias in the surveillance group was analyzed according to the Child-Pugh classification, the surveillance program was found to have had a favorable outcome in Child-Pugh class A patients, but not in Child-Pugh class B/C patients. CONCLUSIONS: HCC surveillance for patients with HCV infection can lead to discovery of tumors at an early stage, especially in Child-Pugh class A, resulting in a favorable outcome.     

Relationship between hepatocellular carcinoma and subtypes of hepatitis C virus: A nationwide analysis

Although hepatitis C virus (HCV) has now been classified into several subtypes, the clinical significance of HCV subtypes is not well known. Typing of HCV is now routinely performed in Japan. In the present study, HCV subtypes in hepatocellular carcinoma (HCC) patients were analysed from nationwide data collected in Japan using a standard questionnaire. Answers to the questionnaire concerning HCV subtypes in patients with chronic hepatitis (CH), liver cirrhosis (LC) and HCC were obtained from 14 hospitals. The prevalence of the 1b-related subtype, which includes the mixed subtype of 1b and 2a or 2b, in patients with LC and HCC in each hospital was higher than in patients with CH, with few exceptions. However, the differences were not statistically significant because of the small number of patients in each hospital. In summarized data from all 14 hospitals, the 1b-related subtype was found in 1370 of 1922 patients with CH (71.2%). In 356 LC and 426 HCC patients, the prevalence of the 1b-related subtype was 79.8 and 80.5%, respectively. The prevalence of the 1b-related subtype in patients with LC and HCC was significantly higher than in patients with CH. There was no significant difference between the prevalence of the 1b-related subtype in patients with HCC and LC. These results indicate that the oncogenic activity of subtype 1b, although not yet clearly characterized, may be stronger than subtypes 2a and 2b.     

Utility of the low-accelerating-dose regimen in 182 liver recipients with recurrent hepatitis C virus

AIM: To describe our experience using a low-acceleratingdose regimen(LADR) with pegylated interferon alpha-2a and ribavirin in treatment of hepatitis C virus(HCV) recurrence. METHODS: From 2003, a protocolized LADR strategy was employed to treat liver transplant(LT) recipients with recurrent HCV at our institution. Medical records of 182 adult patients with recurrent HCV treated with LADR between 1/2003 and 1/2011 were reviewed. Histopathology from all post-LT liver biopsies were reviewed in a blinded fashion. Paired recipient and donor IL28 B status were assessed. A novel technique was employed to ascertain recipient and donor IL28B(rs12979860) Gt data using DNA extracted from archival FFPE tissue from explanted native livers and donor gallbladders respectively. The primary endpoint was SVR; secondary endpoints examined include(1) patient and graft survival;(2) effect of anti-viral therapy on liver histology(fibrosis and inflammation);(3) incidence of on-treatment development of ACR, CDR, or PCH;(4) association of recipient and donor IL28 B genotype with SVR; and(5) incidence of antiviral therapy-associated adverse events(anemia, leukopenia, thrombocytopenia, depression) and hepatic decompensation.RESULTS: The overall SVR rate was 38%(29% Gt1, 67% Gt2, 86% Gt3 and 58% Gt4). HCV Gt(P 0.0001), donor age(P = 0.003), cytomegalovirus mismatch(P = 0.001), baseline serum bilirubin(P = 0.002), and baseline viral load(P = 0.04) were independent predictors for SVR. SVR rates were significantly higher in the recipient-CC/donor-non CC pairs(P = 0.007). Neither baseline fibrosis nor change in fibrosis stage after anti-viral therapy were associated with SVR. Fibrosis progressed in 72% of patients despite SVR. Median graft survival was 91 mo. Five-year patient survival was superior in patients who achieved SVR(97% vs 82%, P = 0.001). Pre-treatment ALP ≥ 150 U/L(P = 0.01), total bilirubin ≥ 1.5 mg/d L(P = 0.001) and creatinine ≥ 2 mg/d L(P = 0.001) were independently associated with patient survival. Only 13% of patients achieving SVR died during the followup period. Treatment discontinuation and treatmentrelated mortality occurred in 35% and 2.2% of patients, respectively. EPO, G-CSF and blood transfusion were needed in 89%, 40% and 23% of patients, respectively. Overall hospitalization rate for treatment-related serious adverse events was 21%. Forty-six(25%) of the patients were deceased; among those who died, 25(54%) were due to liver-related complications, and 4 deaths(9%) occurred while receiving therapy(2 patients experienced hepatic decompensation and 2 sepsis). CONCLUSION: LADR strategy remains relevant in managing post-LT recurrent HCV where access to DAAs is limited. SVR is associated with improved survival, but fibrosis progression still occurs.     

Clinical application of next-generation sequencing for the management of desmoid tumors: A case report and literature review

Rationale:Desmoid tumors are rare myofibroblastic neoplasms characterized by local invasiveness and high rates of recurrence, and sometimes mimic local recurrence of previously resected malignancies. Previous studies have suggested that molecular profiling may be useful for the diagnosis of the tumors and risk stratification. However, the clinical utility of next-generation sequencing (NGS) for the management of desmoid tumors has not been established.Patient concerns:A 42-year-old man visited our clinic for routine follow-up 1 year after left upper lobe lingular segmentectomy for lung adenocarcinoma.Diagnoses:Chest computed tomography showed a pleural mass adherent to the thoracotomy site. Positron emission tomography revealed mildly increased metabolism with a maximal standardized uptake value of 2.7 within the tumor, suggesting local recurrence of the previous neoplasm. Exploratory thoracotomy and en bloc resection of the tumor revealed spindle cells in a massive collagenous tissue consistent with a desmoid tumor.Interventions:NGS was performed to confirm the diagnosis and to identify any genetic alterations that might be relevant to the prognosis of this tumor. The tumor harbored an S45F mutation in CTNNB1, which has been correlated with a high recurrence rate. Therefore, we performed adjuvant radiotherapy on the resection bed at a dose of 56 Gy.Outcomes:The patients experienced no postoperative or radiotherapy-related complications. Periodic follow-up examinations using computed tomography were performed every 3 months, and no evidence of recurrence of either tumor was observed during the 38 months after the last surgery.Lessons:To the best of our knowledge, this is the first case reporting the clinical application of NGS and aggressive treatment based on the genotyping results for the management of a desmoid tumor. Our case highlights the need to consider desmoid tumors among the differential diagnoses when a pleural mass is encountered at a previous thoracotomy site. More importantly, molecular profiling using NGS can be useful for the establishment of a treatment strategy for this tumor, although further investigations are required.