**3. Systemic inflammatory response**

Many tumours, including hepatocellular carcinoma, evoke systemic inflammatory reaction (SIR). In the recent years, cancer-induced SIR has become an attractive research area as changes in blood cell counts or ratios or blood levels of certain proteins are associated with the biological potential, course and treatment response in many malignant tumours. Although complex pathogenesis lies behind these changes, SIR can be evaluated by simple, widely available and economically feasible blood tests.

SIR in cancer patients develops through local and central mechanisms. Locally, the invasive growth of malignant tumour injures surrounding parenchyma, connective tissues and endothelium. The tissue damage leads to inflammation necessitating supply of inflammatory cells from the bone marrow through the circulation. The production of acute phase proteins becomes upregulated as well. Endothelial injury activates platelets; indeed, the association between hypercoagulation and advanced cancers is classical. Tumour necrosis and hypoxia are additional causes of local inflammatory response. Cancer can also evoke immune response manifesting by local cellular reactions. In turn, the inflammation can have both tumour-enhancing and tumour-suppressing outcomes. The released cytokines and transcription factors can upregulate the proliferation of malignant cells. Release of metalloproteinases and other enzymes can promote tissue degradation facilitating invasion. Angiogenesis can be upregulated as well. The immune system, in turn, can limit growth of the tumour.

The systemic effects of cancer include alterations in bone marrow function, especially myelopoiesis. Besides the increased production and release of leukocytes, immature myeloid cells, including the precursors of granulocytes and monocytes, are retained in early stages of differentiation. Immature myeloid cells can act as immune suppressors and generate pre-metastatic niches, among other pathogenetic processes [13]. Thus, it has even been stated that cancer is an inflammatory disease [14]. Further, neutrophils can form neutrophil extracellular traps developed from externalised DNA network. These nets are bidirectionally associated with platelet activation and can contribute to cancer progression via several mechanisms; therefore, neutrophil extracellular traps also represent an attractive treatment target [15].

Cancer-related SIR involves cells of innate and adaptive immunity as well as soluble factors. Macrophages are recruited in tumour by hypoxia and tumour-released molecular agents including growth factors and cytokines [16]. Macrophage phenotype switch from tumoursuppressing classical M1 to tumour-promoting M2 subtype promotes angiogenesis and immunosuppression. Platelet activation contributes to cancer progression and patient mortality [15]. Neutrophils are locally recruited in the cancer via chemokine signalling. Neutrophil activation can contribute to angiogenesis and increased blood vessel permeability locally and metastatic spread systemically. In addition, immature myeloid cells and neutrophil extracellular traps might have tumour-promoting activity. These molecular events also highlight the association between infection or surgery-induced inflammation [17] and cancer relapse or metastatic spread. Thus, innate immunity is generally thought to act as tumour enhancers. In contrast, lymphocytes representing the adaptive immunity are considered to have tumour-suppressing effects [16], although contrary effects have been ascribed to certain subpopulations [18].

HCC can be considered a classical inflammation-induced cancer, as its most common risk factors are hepatitis B and hepatitis C virus infections. Inflammation is also present in liver tissues in patients affected by alcohol-induced hepatitis or NASH. Thus, SIR is not expected to have diagnostic value. Indeed, SIR parameters change before the tumour develops, e.g., increased NLR has been observed in chronic viral hepatitis C [19]. NLR is an independent prognostic factor in liver cirrhosis [20].

Wide variety of inflammation-based markers could be used as indicators of HCC prognosis, tumour recurrence and response to specific treatment. In particular, neutrophil, lymphocyte and platelet counts as well as C-reactive protein and albumin and their combinations, neutrophil-to-lymphocyte ratio (NLR), platelet-to-lymphocyte ratio (PLR) and Glasgow prognostic score (GPS) have prognostic value.

### **3.1. Neutrophil-to-lymphocyte ratio in HCC**

miRNA-30c, miRNA-93, miRNA-122, miRNA-125b, miRNA-130a and miRNA-222) were significantly (p < 0.05) decreased in HCC *versus* chronic viral hepatitis C patients and four (miRNA-93, miRNA-122, miRNA-125b and miRNA-130a) in HCC *versus* non-HCC patients [8]. Panel of three miRNAs (upregulated miRNA-92a-3p, downregulated miRNA-3126-5p and upregulated miRNA-107) could discriminate HCC from healthy controls [9] Evaluating serum levels of 13 miRNAs in HCV-associated chronic hepatitis, liver cirrhosis and HCC *versus* healthy controls, panel of three miRNAs (miRNA-122, miRNA-885-5p and miRNA-29b) in association with serum alpha-fetoprotein (AFP) level could identify HCC *versus* healthy persons, while four miRNAs (miRNA-122, miRNA-885-5p, miRNA-221 and miRNA-22) and AFP were recommended for HCC diagnostics in liver cirrhosis and two (miRNA-22, miRNA-199a-3p) along with AFP—in chronic hepatitis [10]. In another study of HCV-infected patients including cases of HCV-related chronic hepatitis, liver cirrhosis and HCC, serum levels of miRNA-126, miRNA-129, miRNA-155, miRNA-203 and miRNA-223 were significantly decreased in HCC *versus* non-HCC patients [11]. Panel of eight miRNAs was assessed in hepatitis B virus-infected patients diagnosed with HCC or liver cirrhosis as well as in healthy controls. The levels of hsa-miRNA-206, hsa-miRNA-141-3p, hsa-miRNA-433-3p and hsamiRNA-1228-5p were significantly increased in HCC *versus* control group comprising both healthy and cirrhosis patients, while hsa-miRNA-199a-5p, hsa-miRNA-122-5p, hsa-miRNA-

In addition to the diagnostic role, miRNAs have been evaluated in the prognostic aspect. The influence of miRNAs upon HCC stem cells has been exploited. It has also been suggested that

Many tumours, including hepatocellular carcinoma, evoke systemic inflammatory reaction (SIR). In the recent years, cancer-induced SIR has become an attractive research area as changes in blood cell counts or ratios or blood levels of certain proteins are associated with the biological potential, course and treatment response in many malignant tumours. Although complex pathogenesis lies behind these changes, SIR can be evaluated by simple,

SIR in cancer patients develops through local and central mechanisms. Locally, the invasive growth of malignant tumour injures surrounding parenchyma, connective tissues and endothelium. The tissue damage leads to inflammation necessitating supply of inflammatory cells from the bone marrow through the circulation. The production of acute phase proteins becomes upregulated as well. Endothelial injury activates platelets; indeed, the association between hypercoagulation and advanced cancers is classical. Tumour necrosis and hypoxia are additional causes of local inflammatory response. Cancer can also evoke immune response manifesting by local cellular reactions. In turn, the inflammation can have both tumour-enhancing and tumour-suppressing outcomes. The released cytokines and transcription factors can upregulate the proliferation of malignant cells. Release of metalloproteinases and other enzymes can promote tissue degradation facilitating invasion. Angiogenesis can be

upregulated as well. The immune system, in turn, can limit growth of the tumour.

192-5p and hsa-miRNA-26a-5p were downregulated [4].

widely available and economically feasible blood tests.

miRNAs could become treatment targets [12].

50 Hepatocellular Carcinoma - Advances in Diagnosis and Treatment

**3. Systemic inflammatory response**

Neutrophil-to-lymphocyte ratio is the most extensively evaluated parameter of SIR in HCC. NLR, calculated as the ratio between count of neutrophils and lymphocytes in blood, seems to reflect the intensity of systemic pro- and anti-tumour reaction. NLR has shown prognostic importance in surgically treated HCC cases, including early stage tumours; in liver transplantation; sorafenib treatment and different ablation techniques.

Several meta-analyses have been devoted to NLR in HCC patients. Thus, baseline NLR was associated with overall survival and recurrence-free survival, while post-treatment NLR was significantly associated with overall survival [21]. In meta-analysis of NLR in HCC patients treated by liver transplantation, significant association with overall and recurrence-free survival was found [22]. In an early study, NLR was already confirmed as a prognostic factor in HCC. NLR was associated with overall and disease-free survival both generally and after different treatment approaches [23].

2.1, predicted overall survival in solitary small (≤5 cm) HCC after surgical resection and could discriminate outcome in patients having AFP levels not exceeding 400 ng/mL [33]. Further, in a large study of 963 HCC patients treated by potentially curative surgical resection, high NLR (>2.81) was an independent risk factor for overall and recurrence-free survival (both p <   0.001) in the general group as well as in early or intermediate stage HCC: Barcelona Clinic Liver Cancer (BCLC) stages 0/A or B (both p  <  0.05) while no association was found in stage C [34]. NLR was also not associated with early (<1 year) mortality from cancer recurrence after liver resection for huge (at least 10 cm in diameter) HCC in 166 patients [35]. NLR was an independent factor that predicted (p = 0.029) early recurrence after curative resection of HCC presenting as a single focus in 193 Japanese patients [36]. However, contrary results have been reported as well, e.g., NLR had no prognostic significance in early HCC (BCLC stage 0/A)

Innovative Blood Tests for Hepatocellular Carcinoma: Liquid Biopsy and Evaluation of Systemic…

http://dx.doi.org/10.5772/intechopen.76599

53

Somewhat contrasting data are reported regarding NLR in surgically treated patients with more advanced HCC. Although limited significance of NLR was previously noted in highstage or large HCC, some authors have found significant role of NLR in advanced cases. Thus, in 81 hepatectomy-treated HCC patients with portal or hepatic vein tumour thrombosis, high NLR (defined as >2.9) was an independent prognostic factor for worse overall survival, characterised by HR 1.866; 95% CI: 1.048–3.322; p = 0.034. Significant association with recurrencefree interval was found as well. The overall survival in high *versus* low NLR groups was 6.2 months *versus* 15.7 months; p = 0.007, while the recurrence-free survival was 2.2 *versus*

Not only baseline NLR but also the dynamic changes of NLR were found to be important. In 189 patients treated by curative resection for small HCC, the temporal change of NLR was an independent prognostic factor for overall (HR 2.637; 95% CI: 1.356–5.128; p = 0.004) and recurrence-free (HR 2.372; 95% CI: 1.563–3.601; p < 0.001) survival. The 1-, 3- and 5-year survival was 92.7, 70.0 and 53.0% in patients experiencing NLR increase *versus* 96.2, 87.5 and 75.9% in those with decreasing NLR (p = 0.003). High preoperative or postoperative NLR had lower prognostic value in this study [39]. Paralleling observations have been reported by Hung et al., studying patients with HCC recurrence after liver resection. The 5-year post recurrence survival was better (45.9%) in those continuously having NLR ≤ 2.5. Patients who had low NLR (≤2.5) at resection but high level (>2.5) at recurrence had 5-year survival of only

24.6%; the difference was significant as reflected by p = 0.013 [29].

**3.3. Neutrophil-to-lymphocyte ratio in patients receiving transplantation for HCC**

Different aspects of NLR have been evaluated in HCC patients subjected to liver transplantation, including transplantation from living donor. The prognostic value of NLR was revealed already in the early studies. Thus, NLR (applying the cut-off level at 5.0) was an independent predictor of overall and recurrence-free survival in 219 Italian patients after liver transplanta-

By univariate analysis, preoperative NLR was significantly associated by disease-free survival and overall survival in HCC patients after liver transplantation. The 1-, 3- and 5-year overall

treated by surgical resection in 324 patients [37].

3.7 months; p = 0.039 [38].

tion for HCC [40].

#### **3.2. Neutrophil-to-lymphocyte ratio in surgically treated HCC**

In HCC patients subjected to curative resection, several research groups have identified high NLR as an independent, significant predictive factor, associated with worse overall survival [24–27], shorter recurrence-free survival [24, 25, 28] and higher frequency of recurrence [29].

The studies show some variability in design, group size and cut-off level. However, the reported differences in survival are not only statistically significant but also biologically important. In 672 HCC patients treated by surgical resection, the recurrence rate within 1, 3 and 5 years was 77.4, 55.2 and 44.8% in those having low NLR (≤2.5), while the corresponding recurrence rates were 64.1, 45.2 and 35.5% (p = 0.016) in high NLR group [29]. In 303 patients who underwent surgical liver resection for HCC, high NLR (>2.0, based on ROC-detected cut-off) was significantly associated both with shorter recurrence-free and overall survival (both p < 0.001), but multivariate analysis confirmed it as an independent prognostic factor only for overall survival (hazard ratio (HR) 1.724; 95% confidence interval (CI): 1.241–2.394; p = 0.001). Huang et al. evaluated 1659 HCC patients, stratifying them by NLR quartiles. The 5-year overall survival rate was 60% in the lowest quartile contrasting with 27% in the highest quartile. NLR was associated by HR of mortality 1.031; 95% CI: 1.002–1.060; p = 0.033 [30]. In a large Japanese study, enrolling 958 patients who underwent hepatectomy, the 5-year survival rate was 72.9% in low NLR (<2.81) group *versus* 51.5% in patients with high NLR [24]. In another 256 Japanese patients, NLR was confirmed by multivariate analysis as an independent prognostic factor both for overall and recurrence-free interval. The respective hazard ratios were 2.59; 95% CI: 1.56–4.31; p < 0.001 and 2.11; 95% CI = 1.44–3.11; p < 0.001 [25]. In a smaller study of 113 patients, the recurrence-free survival was 42.4 months in patients having NLR < 3 but 7.9 months in those having NLR ≥ 3.0. The respective HR was 2.58; p = 0.002 [28].

The findings in Western patients have been less positive. In the largest Western series comprising 370 patients, treated in Memorial Sloan Kettering Cancer Center in New York, USA, PLR, but not NLR, was independently associated with worse recurrence-free survival and overall survival [31].

NLR retains prognostic value in several subgroups of surgically treated patients, including early cases. By multivariate analysis, preoperative NLR (at cut-off 2.8) was the strongest independent prognostic factor for overall survival after liver resection with curative intent for TNM stage I HCC. The HR was 2.69; 95% CI: 1.57–4.59; p < 0.001. The 5-year survival in high *versus* low NLR group was 45.0 *versus* 76.4%; p < 0.001. Interestingly, the association with survival in stage II or III was not significant, reflected by p = 0.283 and p = 0.155. In stage I patients, high NLR predicted more frequent extrahepatic recurrence (p = 0.006). As growth of HCC is associated with grade progression, these results suggest that NLR reflects the biological potential of HCC [32]. In Chinese cohort of 222 patients, preoperative NLR, using cut-off at 2.1, predicted overall survival in solitary small (≤5 cm) HCC after surgical resection and could discriminate outcome in patients having AFP levels not exceeding 400 ng/mL [33]. Further, in a large study of 963 HCC patients treated by potentially curative surgical resection, high NLR (>2.81) was an independent risk factor for overall and recurrence-free survival (both p <   0.001) in the general group as well as in early or intermediate stage HCC: Barcelona Clinic Liver Cancer (BCLC) stages 0/A or B (both p  <  0.05) while no association was found in stage C [34]. NLR was also not associated with early (<1 year) mortality from cancer recurrence after liver resection for huge (at least 10 cm in diameter) HCC in 166 patients [35]. NLR was an independent factor that predicted (p = 0.029) early recurrence after curative resection of HCC presenting as a single focus in 193 Japanese patients [36]. However, contrary results have been reported as well, e.g., NLR had no prognostic significance in early HCC (BCLC stage 0/A) treated by surgical resection in 324 patients [37].

treated by liver transplantation, significant association with overall and recurrence-free survival was found [22]. In an early study, NLR was already confirmed as a prognostic factor in HCC. NLR was associated with overall and disease-free survival both generally and after

In HCC patients subjected to curative resection, several research groups have identified high NLR as an independent, significant predictive factor, associated with worse overall survival [24–27], shorter recurrence-free survival [24, 25, 28] and higher frequency of recurrence [29]. The studies show some variability in design, group size and cut-off level. However, the reported differences in survival are not only statistically significant but also biologically important. In 672 HCC patients treated by surgical resection, the recurrence rate within 1, 3 and 5 years was 77.4, 55.2 and 44.8% in those having low NLR (≤2.5), while the corresponding recurrence rates were 64.1, 45.2 and 35.5% (p = 0.016) in high NLR group [29]. In 303 patients who underwent surgical liver resection for HCC, high NLR (>2.0, based on ROC-detected cut-off) was significantly associated both with shorter recurrence-free and overall survival (both p < 0.001), but multivariate analysis confirmed it as an independent prognostic factor only for overall survival (hazard ratio (HR) 1.724; 95% confidence interval (CI): 1.241–2.394; p = 0.001). Huang et al. evaluated 1659 HCC patients, stratifying them by NLR quartiles. The 5-year overall survival rate was 60% in the lowest quartile contrasting with 27% in the highest quartile. NLR was associated by HR of mortality 1.031; 95% CI: 1.002–1.060; p = 0.033 [30]. In a large Japanese study, enrolling 958 patients who underwent hepatectomy, the 5-year survival rate was 72.9% in low NLR (<2.81) group *versus* 51.5% in patients with high NLR [24]. In another 256 Japanese patients, NLR was confirmed by multivariate analysis as an independent prognostic factor both for overall and recurrence-free interval. The respective hazard ratios were 2.59; 95% CI: 1.56–4.31; p < 0.001 and 2.11; 95% CI = 1.44–3.11; p < 0.001 [25]. In a smaller study of 113 patients, the recurrence-free survival was 42.4 months in patients having NLR < 3 but 7.9 months in those having NLR ≥ 3.0. The respective HR was 2.58; p = 0.002 [28]. The findings in Western patients have been less positive. In the largest Western series comprising 370 patients, treated in Memorial Sloan Kettering Cancer Center in New York, USA, PLR, but not NLR, was independently associated with worse recurrence-free survival and

NLR retains prognostic value in several subgroups of surgically treated patients, including early cases. By multivariate analysis, preoperative NLR (at cut-off 2.8) was the strongest independent prognostic factor for overall survival after liver resection with curative intent for TNM stage I HCC. The HR was 2.69; 95% CI: 1.57–4.59; p < 0.001. The 5-year survival in high *versus* low NLR group was 45.0 *versus* 76.4%; p < 0.001. Interestingly, the association with survival in stage II or III was not significant, reflected by p = 0.283 and p = 0.155. In stage I patients, high NLR predicted more frequent extrahepatic recurrence (p = 0.006). As growth of HCC is associated with grade progression, these results suggest that NLR reflects the biological potential of HCC [32]. In Chinese cohort of 222 patients, preoperative NLR, using cut-off at

different treatment approaches [23].

52 Hepatocellular Carcinoma - Advances in Diagnosis and Treatment

overall survival [31].

**3.2. Neutrophil-to-lymphocyte ratio in surgically treated HCC**

Somewhat contrasting data are reported regarding NLR in surgically treated patients with more advanced HCC. Although limited significance of NLR was previously noted in highstage or large HCC, some authors have found significant role of NLR in advanced cases. Thus, in 81 hepatectomy-treated HCC patients with portal or hepatic vein tumour thrombosis, high NLR (defined as >2.9) was an independent prognostic factor for worse overall survival, characterised by HR 1.866; 95% CI: 1.048–3.322; p = 0.034. Significant association with recurrencefree interval was found as well. The overall survival in high *versus* low NLR groups was 6.2 months *versus* 15.7 months; p = 0.007, while the recurrence-free survival was 2.2 *versus* 3.7 months; p = 0.039 [38].

Not only baseline NLR but also the dynamic changes of NLR were found to be important. In 189 patients treated by curative resection for small HCC, the temporal change of NLR was an independent prognostic factor for overall (HR 2.637; 95% CI: 1.356–5.128; p = 0.004) and recurrence-free (HR 2.372; 95% CI: 1.563–3.601; p < 0.001) survival. The 1-, 3- and 5-year survival was 92.7, 70.0 and 53.0% in patients experiencing NLR increase *versus* 96.2, 87.5 and 75.9% in those with decreasing NLR (p = 0.003). High preoperative or postoperative NLR had lower prognostic value in this study [39]. Paralleling observations have been reported by Hung et al., studying patients with HCC recurrence after liver resection. The 5-year post recurrence survival was better (45.9%) in those continuously having NLR ≤ 2.5. Patients who had low NLR (≤2.5) at resection but high level (>2.5) at recurrence had 5-year survival of only 24.6%; the difference was significant as reflected by p = 0.013 [29].

### **3.3. Neutrophil-to-lymphocyte ratio in patients receiving transplantation for HCC**

Different aspects of NLR have been evaluated in HCC patients subjected to liver transplantation, including transplantation from living donor. The prognostic value of NLR was revealed already in the early studies. Thus, NLR (applying the cut-off level at 5.0) was an independent predictor of overall and recurrence-free survival in 219 Italian patients after liver transplantation for HCC [40].

By univariate analysis, preoperative NLR was significantly associated by disease-free survival and overall survival in HCC patients after liver transplantation. The 1-, 3- and 5-year overall survival rate in high *versus* low NLR group was 81.3, 56.7 and 51.0 *versus* 90.9, 74.2 and 66.8% (p = 0.041). Similarly, the 1-, 3- and 5-year recurrence-free survival rate in high *versus* low NLR group was 65.3, 48.5 and 39.4 *versus* 80.0, 68.0 and 65.2%. The difference was also significant as reflected by p = 0.013 [41]. Further, NLR was an independent prognostic factor for overall and recurrence-free survival after liver transplantation for HCC as was shown by multivariate analysis of 160 Western patients [42]. NLR was proved to be an independent risk factor for overall survival (p < 0.001) and recurrence-free survival (p = 0.003) in 248 male patients treated by liver transplantation [43]. Harimoto et al. reported on 213 patients receiving living donor liver transplantation for HCC. High preoperative NLR ≥ 2.66 was an independent predictor of recurrence [44].

**3.5. Neutrophil-to-lymphocyte ratio in HCC patients undergoing tumour ablation**

independent prognostic factor for survival after TACE [56].

overall survival were associated with NLR [59].

confirmed by p < 0.001 [61].

pendent predictor of worse survival [65].

**3.6. Neutrophil-to-lymphocyte ratio and tumour characteristics**

NLR has been investigated in regard to different embolisation and ablation techniques. High NLR (>3) predicted significantly worse treatment results (p = 0.014) and early disease progression (p < 0.0001) in 86 treatment-naive patients subjected to arterial chemoembolisation or radioembolisation [55]. Elevated pre-treatment NLR (>1.85) was associated with overall survival and disease-free survival in 178 HCC patients subjected to transcatheter arterial chemoembolisation (TACE). The median survival in high *versus* low NLR group was 8 *versus* 17.5 months. The 1-, 3- and 5-year overall survival rates in these groups were 42.1, 19.6 and 9.5 *versus* 57.3, 44.1 and 27.2%, respectively (p < 0.001). Differences in disease-free survival were significant as well (p < 0.001). Multivariate analysis confirmed NLR as a significant (p = 0.04),

Innovative Blood Tests for Hepatocellular Carcinoma: Liquid Biopsy and Evaluation of Systemic…

http://dx.doi.org/10.5772/intechopen.76599

55

In patients with advanced HCC treated by hepatic arterial infusion chemotherapy, high NLR was a significant predictor of lower response rate, worse progression-free and overall survival [57]. Baseline NLR was a significant predictor of treatment response and progression-free survival after hepatic arterial infusion chemotherapy for advanced HCC [58]. In patients receiving hepatic arterial infusion chemotherapy by cisplatin and fluorouracil, response rate and

Dynamic changes of NLR had independent prognostic significance (p = 0.035) in HCC with portal vein tumour thrombosis treated by microwave ablation after transarterial chemoembolisation [60]. In 506 patients treated by thermal ablation of recurrent HCC, high pretreatment NLR (≥2.14) was a prognostic factor for recurrence-free survival, confirmed by Cox multiple regression analysis. The 1- and 3-year recurrence rates in high *versus* low NLR groups were 57.9 and 82.5 *versus* 20.7 and 31.6%. The difference was statistically significant,

Pre-treatment NLR was associated with worse overall survival in early HCC after radiofrequency ablation. Post-treatment NLR was associated both with worse overall survival and recurrence in early HCC after radiofrequency ablation [62]. Similarly, NLR dynamics, but not pre-treatment NLR, was an independent prognostic factor for overall survival and recurrencefree survival in patients with small HCC treated by radiofrequency ablation [63]. In patients treated by radiofrequency ablation for HCC, post-treatment NLR was associated with recurrence and survival. Pre-treatment NLR was associated with recurrence only in patients who had HBV infection and HCC but not in those who developed HCC in association with HCV infection [64]. In unresectable HCC treated by radioembolisation, elevated NLR was an inde-

NLR has been mostly assessed in correlation with survival or treatment response. However, some observations are reported on the association between systemic inflammatory response and tumour morphology in gross and microscopic level. Thus, high NLR is observed in patients having larger tumours, multiple HCC foci, higher grade of HCC and vascular invasion [29]. In few studies, the infiltration of neutrophils and macrophages in liver tumours has been assessed. Peritumoural tissues are characterised by higher ratio between neutrophils

NLR can be used in prognostic models to identify patients who exceed Milan criteria but still have good overall and tumour-free survival. In study reported by Wang et al., male patients were enrolled and thus the proposed models were verified in males only [43]. Combination of NLR and Hangzhou criteria has been suggested to identify patients who can be successfully treated by liver transplantation [45]. NLR has been included in the MORAL scores to predict recurrence after liver transplantation, and these scores were superior to Milan criteria [46]. Complex evaluation of NLR along with fibrinogen increases the prognostic accuracy in order to predict disease-free survival and overall survival in HCC patients treated by liver transplantation [41]. Pretransplantation NLR along with levels of C-reactive protein has been combined with Milan criteria to develop new selection criteria for living donor liver transplantation beyond Milan criteria [47].

However, contrasting findings have been published as well. Thus, NLR did not predict posttransplantation recurrence or worse overall survival in 150 patients within Milan criteria [48]. Limited prognostic impact of NLR was found in 124 patients who underwent living donor liver transplantation [49]. In Western patients, NLR was not predictive of treatment success regarding liver transplantation or other tested approaches (hepatectomy, transarterial chemoembolisation). Although the group was quite small (75), Child-Pugh and Model for End Stage Liver Disease (MELD) scores were informative [50].

### **3.4. Neutrophil-to-lymphocyte ratio in sorafenib-treated HCC patients**

In patients with unresectable HCC, treated by multikinase inhibitor sorafenib, high NLR (>3.1) was a significant independent prognostic factor, associated with worse overall survival. Better treatment response was observed in patients with low NLR [51]. The findings were confirmed by another study, reporting on 442 sorafenib-treated patients (Japan, Italy and United Kingdom) with advanced HCC. High NLR again was an independent prognostic factor, predicting shorter survival with HR 1.218; 95% CI: 1.108–1.322; p < 0.0001 [52].

Regarding combined approach, high pre-treatment NLR (>3.0) was an independent predictor of worse overall survival in 40 patients with unresectable HCC treated by transcatheter arterial embolisation and sorafenib. The median survival in high *versus* low NLR group was 14 months (95% CI = 10.1–17.9) *versus* 26 months (95% CI = 17.4–34.6). The difference was significant (p = 0.001) and biologically remarkable [53]. Other researchers have also confirmed that NLR was independent predictor of overall survival in patients with advanced HCC treated by sorafenib [54].
