Increase of non- HBV, non- HCV related hepatocellular carcinoma -Over 20 years single institution study-Yuko

Background: We previously reported the trends of etiologies of hepatocellular carcinoma (HCC) between 1995 and 2009. By updating this study, we aimed to evaluate the incidence of HCC with non- hepatitis B viral and non- hepatitis C viral (NBNC) etiologies and explore the clinical characteristics, also. Methods: Consecutive 2171 HCC patients at our institution were enrolled between 1992 and 2018 in this retrospective cohort study. A comparative study was conducted by dividing into two groups, an early group from 1992 to 2009 and a late group from 2010 to 2018. Results: NBNC-HCC account for 514 patients (23.6%). The proportion of NBNC-HCC has continued to increase from 26.5% in 2009 to 46.3% in 2018. NBNC-HCC were getting older (median ages, 67 to 73 years). Type 2 diabetes mellitus (48.5% to 60.3%: P=0.008), hypertension (48.5% to 57.4%: P=0.047), and hyperlipidemia (39.2% to 53.8%: P=0.001) has increased signicantly in recent years. The median FIB-4 index have decreased (4.37 to 3.61: P=0.026) and the median platelet counts have increased (15.1 to 17.9: P=0.013) signicantly. Among 514 NBNC-HCC, 194 patients underwent hepatic resection in which 14.9%, 29.3%, and 55.8% were based on nonalcoholic steatohepatitis (NASH), alcoholic liver disease (ALD), and cryptogenic, respectively. Cirrhosis was detected in 72.4%, 38.6%, and 15.5% of NASH, ALD and cryptogenic, respectively. The prevalence of cirrhosis in NASH were signicantly higher than other groups (P<0.001), overall, 70% of the background liver of NBNC-HCC was not cirrhosis. On the other hands, the median FIB-4 index in cryptogenic HCC were 2.56 and signicantly


Background
Hepatocellular carcinoma (HCC) is one of the most common malignant tumors in the world. In Japan, it ranks fth in men and sixth in women as a cause of death from malignancies [1] and 93.3 % of primary liver cancers are HCC [2]. Until now, hepatitis C virus (HCV) and hepatitis B virus (HBV) has contributed to primary liver cancer, especially the development of HCC. That is, HCV-related HCC account for 67 % of all HCCs followed by HBV-related HCC that account for 16 % of all HCC in the vast majority of such cases worldwide [3].
However, because of efforts to control HBV and HCV infections, mortality rates have started to decrease in Asia and Southern Europe [4]. In those with active chronic HBV infection, long-term suppression using nucleotide analogs may reduce the incidence of HBV-related HCC [5,6], and the eradication of HCV by antiviral therapy can reduce HCV-related HCC [7].
According to our previous report [8], 17% and 67% of HCC were related to HBV-HCC and HCV-HCC, respectively. Fifteen percent of that was negative for hepatitis B surface antigen (HBsAg) and antibody to HCV (HCVAb) (NBNC-HCC). Especially, the incidence of HCV-related HCC has gradually decreased in recent years, whereas the incidence of HCCs associated with NBNC-HCC has gradually increased from 10 % in 1995 to 19 % in 2009 [8]. Furthermore, Tateishi et al. reported that with NBNC-HCC has increased from 10 % in 1991 to 32.5 % in 2015 [9,10].
In addition to alcoholic liver disease (ALD), which was reported as risk factors previously, it is recently becoming known that nonalcoholic fatty liver disease (NAFLD).
Recently, impact of NAFLD or non-alcoholic fatty steatohepatitis (NASH) as the hepatic manifestation of diabetes and obesity has been clearly linked to progression of liver brosis, cirrhosis and HCC risk in several studies [14][15][16]. Recent evidence indicates that obesity and T2DM are HCC risk factors [17][18][19][20]. In Japan, overweight and obese indicates a relative risk of 1.74 compared with normal and low-weight individuals [21]. Moreover, many studies have reported that T2DM is the risk of HCC, revealed a 2-4-fold greater risk of HCC among patients with T2DM compared with nondiabetic controls [22][23][24][25]. Insulin resistance (IR) associated with metabolic syndrome, T2DM and NAFLD leads to increased release of free fatty acids from adipocytes and the release of multiple pro-in ammatory cytokines including tumor necrosis factor alpha (TNF-a), interleukin-6 (IL-6), leptin, and resistin, as well as decreased amounts of adiponectin, and obesity is characterized by a low-grade chronic in ammatory response that is associated with increased in HCC [14,[26][27][28][29]. T2DM, obesity, and the metabolic syndrome have been growing in prevalence and are independent risk factors for the development of HCC. In this way, many studies have been reported on the risk of NBNC HCC.
These characteristics were similar to our previous report [8], the transition and risk factors in NBNCHCC were examined.
On the other hands, whereas various causes in uence the development of NBNC-HCC, few reports have examining characteristics such as background liver using pathological specimens obtained by hepatectomy. Several studies have reported that NASH can progress to cirrhosis, advanced brosis are important risk factors for HCC [16, [30][31][32], and the prevalence of cirrhosis in the ALD-HCC was signi cantly higher, too [11,17,[33][34][35][36]. NASH and ALD have now become the leading indications for liver transplantation such as HCV and HBV. and investigated the clinical characteristics of NBNC-HCC by employing liver specimens pathologically because of urgent needs for the prevention and the future strategies against NBNC-HCC.

Patients
We retrospectively reviewed 2171 consecutive patients of primary HCC diagnosed at Hiroshima University Hospital from January 1992 to December 2018. All subjects gave informed consent to participate in the study according to the process approved by the ethical committee in Hiroshima University Hospital and conforming to the ethical guidelines of the Declaration of Helsinki.

Laboratory Evaluation
We collected the detailed clinical data (age, gender, history of habitual alcohol consumption, height, weight, lifestyle-related diseases, and laboratory parameters).
Biochemical data were measured using standard techniques for each patient at the time of diagnosis of primary HCC. Fibrosis (FIB)-4 index were calculated as a surrogate marker of liver brosis [37]. FIB-4 index HBV-HCC was de ned as positive for HBsAg and negative for HCVAb, HCV-HCC was de ned as positive for HCVAb and negative for HBsAg, NBNC-HCC was de ned as negative for both HBsAg and HCVAb.
Alcohol liver disease (ALD) was determined when alcohol intake was more than 60 g/day for ve years [39]. The body mass index (BMI) was calculated as body weight in kilograms divided by the square of the height in meters (kg/m 2 ). The HbA1c (%) value was estimated as a National Glycohemoglobin Standardization Program (NGSP) equivalent value (%) calculated by using the formula: A1c (%) = A1c (Japan Diabetes Society (JDS)) (%) + 0.4 %, considering the relational expression of A1c (JDS) (%) measured by the previous Japanese standard substance and measurement methods and HbA1c (NGSP) [40]. Type 2 diabetes mellitus (T2DM) was diagnosed in cases with a high fasting plasma glucose (FPG, >126 mg/dL), high HbA1c (NGSP) (HbA1c, >6.5%), use of glucose-lowering agents, or a self-reported history of a clinical diagnosis.
Hyperlipidemia was diagnosed when the patient was being treated with lipid-lowering medications or had elevated levels of total cholesterol higher than 220 mg/dL and/or the triglyceride level was over 150 mg/dL. Hypertension was diagnosed when the patient was on antihypertensive medications and/or had a resting recumbent blood pressure of 130/85 mmHg or more on at least two occasions.

Pathological evaluation
The pathological status of underlying liver disease was based on microscopic examination of the noncancerous part of the surgical specimen or biopsy specimen with hematoxylin-eosin and Azan staining.
The diagnosis of HCC was based on the hypervascular staining pattern of the arterial phase and the hypovascular staining pattern of the portal phase, and was con rmed by dynamic CT, magnetic resonance imaging, and/or angiography [43]. Tumors without enhancement upon imaging were diagnosed by ne-needle biopsy. Clinical staging (TNM classi cation) was performed according to the criteria of the Liver Cancer Study Group of Japan [44].

Statistical analysis
Results are shown as median values or percentages. Continuous variables are reported as median deviation and were compared using the Mann-Whitney U-test. Categorical variables were compared using the chi-square or Fisher exact test, as appropriate. The Cochran Armitage trend test was used to evaluate increasing or decreasing trends. All statistical analyses were performed using the SPSS software package (version SPSS 23.0, IBM, Armonk, NY, USA). A P value <0.05 was considered a statistically signi cant difference.

Trend of etiology of liver diseases in patients with HCC
Among 2171 patients with primary HCC from 1992 to 2018, 361 patients (16.6%) and 1271 patients (58.5%) were related to HBV-HCC and HCV-HCC, respectively (Fig.1). Five hundred and fourteen patients (23.6%) of that was negative for HBsAg and HCVAb (NBNC-HCC). Twenty-ve (1.1%) patients with HCC were positive for both HBsAg and HCVAb (BC-HCC). In order to compare with the previous reports up to 2009, the study was divided into three periods.

Characteristics of patients with NBNC-HCC
Previously, we reported up to 2009 [9], we compared with the characteristics of patients with NBNC-HCC from 2010 to 2018 by period ( Table 2).
Patients with NBNC-HCC during 2010-2018 were signi cantly older than other groups (P<0.001). As mentioned in the previous term, rate and number of NBNC-HCC tended to increase, accordingly, the number of patients with metabolic syndrome tended to increase, also.

Comparison of FIB-4 index by age in NBNC-HCC patients
In addition, we assessed the FIB-4 index in NBNC-HCC patients by age. The median FIB-4 index indices in subjects from 20 to 39 years, from 40 to 59 years, from 60 to 79 years, and from 80 to 99 years were 1.43, 4.25, 3.97, and 4.39, respectively. There was no signi cant difference in FIB-4 index for age strati cation over 40 years. (Fig.2).

Clinical characteristics of NBNC-HCC patients with hepatic resection
Among 514 patients with primary NBNC-HCC, we studied clinical features of 194 patients who underwent hepatic resection (less number of autoimmune hepatitis or primary biliary cholangitis were excluded). We assessed the histological features of non-tumorous liver in NBNC-HCC patients. Here, in this study, we de ned cases in which NASH or ALD could not clearly diagnosed in advance based on pathological results or medical history as cryptogenic.
The median age in cryptogenic group was signi cantly older than NASH groups (P=0.018). The percentage of males was signi cantly higher in ALD and cryptogenic groups than in NASH group (P<0.001).
The median platelet counts in cryptogenic group were signi cantly higher than in other groups (vs NASH; P=0.025, vs ALD; P=0.023) and FIB-4 index in cryptogenic group were signi cantly lower than in other groups (vs NASH; P<0.001, vs ALD; P=0.002). No signi cant differences in other clinical characteristics were observed ( Table 3).

Discussion
In the present study, we retrospectively assessed the clinical characteristics of HCC. The rate and the number of NBNC-HCC tended to increase, whereas those of HCV-HCC tended to decrease especially in recent years. Five hundred and fourteen patients (23.6%) of HCC were NBNC-HCC in 1992-2018. Patients with NBNC-HCC were signi cantly older. The rates of heavy alcohol consumption, T2DM, hypertension, and hyperlipidemia were signi cantly higher than those in other groups. Furthermore, in NBNC-HCC patients, BMI and the platelet counts were signi cantly higher and FIB-4 index were signi cantly lower than other groups.
In accordance with previously reported ndings, our ndings were in agreement with those that people with obesity, T2DM or heavy alcohol consumption and metabolic syndrome are at an increased risk of developing HCC [45,46]. As previously reported [8], these complications are likely to become HCC risks in the future and need attention. Table 2, the patients with NBNC-HCC during 2010-2018 were signi cantly older, and the rates of T2DM, hypertension, and hyperlipidemia were higher than those from 1992 to 2009. The increase in metabolic syndrome is suggested to affect the increase of NBNC-HCC. Furthermore, the platelet counts were higher and FIB-4 index were lower than those from 1992 to 2009. This might be because HCC occurred even in patients with non-advanced liver brosis. These observation shows us that NBNC patients need to be aware that HCC develops independently of brosis stage. Although it is suggested that the systemic metabolic syndrome environment may cause a systemic in ammatory condition and this might be associated with thrombocytosis, the reason why platelets are maintained more than before has not been clari ed in this study, and awaits future study. FIB-4 index tended to be higher in the elderly, but there is no signi cant difference by age as shown in Fig.2. Because of small number of young patients with HCC, it is unsure but FIB-4 index might become a useful screening index of HCC.

As shown in
The TNM stage in NBNC-HCC was signi cantly advanced more than that in other groups. This is because patients with NBNC-HCC do not receive regular HCC surveillance whereas HBV-HCC and HCV-HCC patients received regular HCC surveillance. HBV and HCV infection related to long-term infection and the progression of liver brosis often leads to chronic, hepatitis, cirrhosis, cirrhosis-related complications, and HCC [47], therefore, HBV and HCV patients received regular surveillance. Similar results have been reported in our past reports [8][9][10], it is necessary to recommend to receive regular HCC surveillance using ultrasonography and/or other available modalities even if patients don't have liver disease such as viral hepatitis, since still so many NBNC-HCC is detected in an advanced stage. Moreover, it is also important to perform screening using CT and MRI with new imaging and scoring system, we think it is necessary to diagnose HCC earlier. [48,49]. Therefore, in order to diagnose HCC at an early stage, it is necessary to use FIB-4 index together.
From the examination of histologically diagnosed background liver, cirrhosis was detected in 72.4%, 38.6%, and 15.5% of patients with NASH, ALD and cryptogenic, respectively. The prevalence of cirrhosis in NASH were signi cantly higher than that in other groups, and cryptogenic had a lower rate of advanced brosis. Until now, many studies have reported that NASH can progress to cirrhosis [50][51][52][53], the severity of liver brosis must be estimated to determine the surveillance and for treatment of NAFLD. In patients with cryptogenic, platelet counts tended to be higher, FIB-4 index was lower, and brosis was not severe. At present, FIB-4 index, one of the recommended scoring systems, uses a cutoff value of 2.67 as a progression of brosis. The median FIB-4 index in cryptogenic was 2.56 and this is similar to the value that has been regarded as an index that increases the risk of developing HCC [54,55]. From these results as well, the FIB-4 index could contribute to HCC screening.
In our study, patients with cryptogenic HCC accounted for the majority of NBNC-HCC. Most (93.5%) of those in cryptogenic patients had any of T2DM, hypertension, hyperlipidemia, obesity, and HBc antibody.
Considering the above, it is possible that HCC progressed without being diagnosed with NASH or Alcoholic liver disease during the course. In the future, patients with metabolic syndrome will need to pay attention to HCC development.
Limitations of our study are that not all the pathological aspects of background liver were available in NBNC-HCC group and that the study population may not re ect the entire patients with HCC in Japan.

Conclusions
The present study elucidated that NBNC-HCC has gradually been increasing in recent years culminating in 40% of all HCC patients. The presence of metabolic syndrome were important risk factors for NBNC-HCC. Furthermore, only advanced brosis in NBNC-HCC are not the determinant for increased HCC risk, and HCC from non-cirrhosis are not uncommon. However, FIB-4 index tends to be higher, considered as one of useful screening of HCC. γ-GTP, γ-glutamytranspeptidase; NASH, non-alcoholic steatohepatitis; FIB-4, brosis-4; ALBI grade, albumin-bilirubin grade; NS, not signi cant.  We assessed the FIB-4 index in NBNC-HCC patients by age. In these box-and-whisker plots, lines within the boxes represent median values; the upper and lower lines of the boxes represent the 75th and 25th percentiles, respectively; the upper and lower bars outside the boxes represent the 90th and 10th percentiles, respectively. NS, not signi cant