Alpha-Fetoprotein and Neutrophil-to-Lymphocyte Ratio: Comparative Analysis in Hepatocellular Carcinoma Diagnosis

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availability of treatment modalities like surgery and transplantation for HCC at an early stage, the outcome has improved to more than 70% [6].Therefore, for better clinical outcomes, diagnosing HCC at an early stage is important.AFP is a very widely used biomarker for HCC worldwide.HCC size, differentiation, invasion, and metastasis are all linked to serum AFP [7].
Values more than 400 ng/ml, although not observed in all cases, are considered diagnostic for HCC [8].Serum AFP is not always reliable because it is often not above the value used for diagnosing HCC in some of the early-stage or late-stage patients with HCC [9].Normal AFP levels are seen in 15-30% of cases of advanced-stage HCC.It has been seen that if a lower cutoff is used.Also, AFP levels are increased in benign diseases of the liver like hepatitis or cirrhosis [10].Therefore, new markers are desirable for early detection of HCC.AFP is only used as a reference biomarker in the early stages of HCC diagnosis when no other clinical information is available.
The progression of tumor is closely linked to inflammatory factors.Inflammatory signaling pathways and tumor microenvironment changes are linked to hepatitis, cirrhosis, and HCC staging [11].There are several inflammatory factors that reflect that a standard inflammatory response is present.And easily obtained at a low-cost examination done routinely [5].NLR has gained attention because of its medical application in several diseases e.g.liver fibrosis, colorectal cancer, and cervical carcinoma [11].Only a few studies have explored the potential of the neutrophil-to-lymphocyte ratio (NLR) for the early detection of hepatocellular carcinoma (HCC), suggesting its potential utility in this context.This observation sought to assess the diagnostic efficacy of combining alpha-fetoprotein (AFP) with NLR in comparison to AFP alone for detecting HCC.

METHODS
Between July 2021 and June 2022, a case-control study was conducted at a tertiary care hospital in Dehradun, Uttarakhand, India.The study received approval from the ethics committee of Swami Rama Himalayan University in Dehradun, India (SRHU/HIMS/ETHICS/2022/339, dated 25/05/2021), and adhered to the guidelines outlined in the Declaration of Helsinki.Written informed consent was obtained from all participants involved in the study.The study enrolled sixty HCC patients aged eighteen and above who had recently been diagnosed and had not yet initiated treatment [2].Patients with concomitant sepsis or other coexisting malignancies were excluded from the study.Controls comprised an equal number of age-and sex-matched individuals with chronic liver disease but without hepatocellular carcinoma (HCC).
Demographic data, including age, sex, place of residence, and socioeconomic status, were collected for each enrolled patient.Detailed medical histories, including alcohol consumption habits, were obtained.Comprehensive clinical assessments were conducted.Both cases and controls underwent a battery of tests and assessments for viral markers.Imaging studies for HCC diagnosis, such as whole-abdominal ultrasonography and contrast-enhanced computed tomography scans (CECT), were performed on all patients.Serum alpha-fetoprotein (AFP) levels, neutrophil and lymphocyte counts, along with other pertinent parameters, were assessed.
The neutrophil-to-lymphocyte ratio (NLR) was computed by dividing the neutrophil count by the lymphocyte count.
Data analysis was conducted using SPSS version 22.0.The Kolmogorov-Smirnov test was employed to assess the normality of quantitative data.Normally distributed quantitative data were presented as mean (±SD), while non-normally distributed data were reported as median and range.The Student's t-test was used for normally distributed quantitative data, and Fisher's exact test (chi-square test) was used for qualitative data comparison.The diagnostic performance of AFP, NLR, and their combination for HCC diagnosis was evaluated using the concordance (C-) statistic, akin to the area under the receiver operating characteristic curve (AUC).Receiver operating characteristic (ROC) curves were utilized to determine accuracy, sensitivity, specificity, and cut-off values for NLR and AFP.Statistical significance was determined with pvalues below 0.05.

RESULTS
Demographic selection of patients with HCC, as well as chronic liver disease (CLD), are depicted in Table 1.The mean age of cases was 57.2±11.54years and of controls 57.01±12.13years.Both among cases and controls, 44 subjects were less than sixty years of age.While 42 subjects were male, and 18 subjects were female.HCV was found to be the most frequently associated with HCC in the current study, with a 53.3% positivity rate.13.3% of the participants had positive hepatitis B surface antigen (HBsAg) results.The C-statistic for NLR in diagnosing HCC was 0.623 (p=0.101,95% CI 0.48-0.77),indicating no statistical significance.Using the optimal cut-off of 4.4, sensitivity and specificity for HCC diagnosis were 63.3% and 60%, respectively.The laboratory parameters of patients with HCC are listed in Table 3.In contrast, AFP demonstrated a statistically significant C-statistic of 0.941 (p=0.000,95% CI 0.88-1.00),with sensitivity and specificity of 93.3% and 83%, respectively, at the optimal cut-off of 8.4.When combined, AFP and NLR yielded a statistically significant Cstatistic of 0.95 (p=0.000,95% CI 0.89-1.00).For HCC diagnosis, sensitivity and specificity were 100% and 86.7%, respectively (Fig. 1).The disparity in C-statistics between AFP and NLR was statistically significant (p=0.0007).
However, the contrast in C-statistics between the combination of AFP and NLR and AFP alone did not reach statistical significance (p=0.54) (Fig. 2).

DISCUSSION
The incubation period is long, and it has a rapid course of development.HCC patients have a high mortality rate [5].With the availability of treatment modalities like surgery and transplantation for HCC at an initial stage, the outcome has improved [6].Therefore, for better clinical outcomes, diagnosis is important.
In a study by Ding et al., patients with HCC had an average age of 47.3 years, with a standard deviation of 13.7 years.The study found that 75.8% of participants were male, while only 24.2% were female [12].Petrick et al. noted that incidence rates among men are two to four times higher than among women [14].Similarly, Kumar reported that in India, men are affected by HCC four times more frequently than women, with the typical age of presentation falling between 40 to 70 years old [15].Consistently, our study also observed a male predominance (2.33:1) and a mean age of 57.2±11.54years among patients diagnosed with HCC, aligning with previous research findings.
Alcohol is the common cause of CLD, which makes up 30% of HCC cases and HCC-associated deaths globally [16].In epidemiological research, smoking is one of the highlighted risk factors for HCC [17].In our study, among cases, 40% were habituated to alcohol, 26.7% had a history of smoking and 46.7% had a history of tobacco chewing.
However, in our study, HCV positivity was seen in more than half of the cases while among controls, two-thirds were non-reactive for viral markers.This difference was found to be statistically significant.HCV was found to be the most frequent cause of HCC in the current study, with a 53.3% positivity rate.13.3% of the participants had positive HBV results [18].
Elevated baseline NLR often signifies systemic and localized inflammation, which promotes tumor invasion and metastasis [25].NLR represents a systemic immunological state that favors tumor invasion while decreasing host immune surveillance, in addition to a microenvironment that is favorable to tumors [26].Various studies show the impact of NLR on overall survival of HCC patients.The association between NLR and HCC was initially explained by Halazun et al., who showed that patients undergoing transplantation of liver for HCC were more likely to experience high recurrence and poor overall survival when their NLR was elevated (>5) [27].
Kayadibi et al (2014) studied whether a higher NLR is linked to tumor recurrence and suggested that though NLR is easily available, it would be better to use it in addition to other inflammatory markers for predicting tumor microenvironment [28].These studies indicate that NLR may help in screening HCC.However, a limited number of studies have evaluated the value of NLR for early diagnosis of HCC and it has been seen that NLR may be useful in this regard.
In our evaluation of HCC diagnostic tools, we examined the utility of AFP and NLR, and compared the recorded alone value of serum AFP with that of serum AFP plus NLR combined.
Our findings revealed that serum AFP displayed a commendable accuracy (AUC=0.941) in detecting HCC and exhibited significantly higher in patients diagnosed with HCC.When utilizing the certain cut-off of 8.4, the sensitivity and specificity for HCC diagnosis were 93.3% and 83%, respectively.
The C-statistic for NLR in diagnosing HCC was 0.623 (p=0.101,95% CI 0.48-0.77).Utilizing the optimal cut-off of 4.4, the sensitivity and specificity for diagnosing HCC were 63.3% and 60%, respectively.While NLR showed a slight elevation in patients with HCC, it did not demonstrate significant accuracy in HCC detection.Additionally, AFP outperformed NLR in diagnosing HCC.These findings align with a study by Johnson et al., where they evaluated the impact of baseline on the overall survival of HCC patients and compared NLR levels between patients with chronic liver disease (CLD) and those with HCC.Although NLR was higher in HCC patients compared to controls at baseline (2.79 vs 2), the difference in NLR values between the two groups was not clinically significant.Furthermore, the accompanying AUC calculation indicated that NLR had very weak diagnostic ability, with an AUC of 0.65 (95% CI 0.62, 0.69) [29].
Study conducted by Ding et al., which investigated the combined value of an inflammatory score and AFP for diagnosing HBV-related HCC, it was found that AFP in conjunction with ALT, AST, and NLR improved the diagnostic accuracy for HBV-HCC compared to individual biomarkers [12].Hu et al. (2019) explored the significance of the platelet ratio (GPR) and neutrophil-to-lymphocyte ratio (NLR) in patients diagnosed with hepatocellular carcinoma (HCC).Their study revealed that pre-intervention hematologic parameters (NLR and GPR) were correlated with the Barcelona Clinic Liver Cancer (BCLC) stages.Integrating these parameters with AFP levels could aid in the early detection of HCC [30].In our study, although not statistically significant, we observed that the combination of AFP and NLR showed a marginal improvement in identifying HCC compared to AFP alone.Consequently, NLR alone does not contribute significantly to HCC diagnosis, but when used alongside AFP, it may modestly improve diagnostic accuracy.

CONCLUSION
NLR, or Neutrophil-to-Lymphocyte Ratio, was not deemed effective for the early diagnosis of hepatocyte carcinoma (HCC).Conversely, Alpha-Fetoprotein (AFP) emerged as a dependable diagnostic marker for HCC.Nevertheless, when AFP was integrated with NLR, their synergistic effect resulted in the highest diagnostic accuracy.Consequently, this amalgamation presents a promising avenue for facilitating the early detection of HCC, thereby enabling timely interventions and potentially improving patient outcomes.