A Combination of Blood Lymphocytes and AST Levels Distinguish Patients with Small Hepatocellular Carcinomas from Non Cancer Patients

Brian I. Carr (  brianicarr@hotmail.com ) Inonu University School of Medicine: Inonu Universitesi Tip fakultesi https://orcid.org/0000-00026111-5077 Harika Gozukara Bag Inonu University Volkan Ince Inonu Universitesi Sami Akbulut Inonu University Veysel Ersan Inonu University Sertac Usta Inonu University Burak Isik Inonu Universitesi Zeki Ogut Inonu Universitesi Adem Tuner Inonu University Sezai Yilmaz Inonu Universitesi


Introduction
Most hepatocellular carcinoma (HCC) patients are diagnosed at an advanced tumor stage, when surgical therapies cannot be used (1). In our experience and that of others, only a minority of cases can currently be treated by surgery (2), which is the main treatment that results in long survival. Although some larger HCCs can be treated by resection, survival post-surgery is signi cantly less than for smaller size HCCs (3).
Thus, tumor size is prognostically important. Given the importance of small HCC size in HCC treatment outcomes, emphasis has been placed on screening of patients at risk, such as those with chronic hepatitis B or C. Nevertheless, surveillance programs can result in diagnosis of smaller HCCs with increased survival (4,5). Thus, screening is important, considering the estimated global 905,677 new HCC cases annually, and 830,180 deaths (6). There is thus a need for simple, cheap and reliable tumor markers to be used at patients at risk of developing HCC, to diagnose smaller size HCCs, where surgical therapy is most effective. We report here a comparison of clinical laboratory parameters in patient with small HCCs and in patients without known HCC, to evaluate differences that might have a possible future use in screening.

Clinical methods
Patients having live donor liver transplantation for HCC (n=101) or non-tumor (n=275) liver disease indications (predominantly chronic hepatitis B) at our Liver Transplantation Institute are the subjects of this study. The data were analyzed retrospectively. This study was approved by Inonu University Institutional Review Board (Approval no: 2021/2544). Patients had either a small HCC ≤3cm or no tumor as judged by pre-transplant CAT scan evaluation. All patients had baseline radiological evaluation, complete blood counts and standard liver function tests.

Statistical methods
Normal distribution of the quantitative data was evaluated by Kolmogorov-Smirnov test. Quantitative data were summarized by median, minimum and maximum values. Mann-Whitney U test was used to compare two independent groups. Qualitative data was expressed as count and percentage. Pearson's chi-square, continuity corrected chi-square or Fisher's exact tests were used for comparisons where appropriate. Odds ratio (OR) estimations were obtained by both univariate and multivariate binary logistic regression analysis. Signi cance level was considered as 0.05 in all analyzes.

Results
Comparison of blood parameters for non HCC and HCC patients A comparison was made of the common peripheral blood count and liver function test parameters between patients with small HCCs (n=101) versus patients with hepatitis or cirrhosis (n=275) who did not have an HCC diagnosis on pre-transplant evaluation. There were statistically signi cant differences between groups in terms of lymphocytes (p<0.001) and AST (p<0.002) levels (Table 1) using both univariate and multivariate logistic regression analyses. Abbreviations: WBC, white blood count (x10 9 /L); Lymphs, lymphocytes (x10 9 /L); T. bil, total bilirubin (mg/dL); AST, aspartate amino transferase; (IU/mL) ALT, alanine aminotransferase (IU/mL); Eosinophils (x10 9 /L).

Two-parameter models
We next built a 2-parameter model, based upon the only 2 parameters that were individually signi cantly different in Table 1, namely blood lymphocyte and AST levels. The median values of lymphocytes and AST for the non HCC patients were used for the dichotomization (  Abbreviations: Lymphs, lymphocytes (x10 9 /L); AST, aspartate amino transferase (IU/mL).
HCC tumor and blood characteristics using the 2-parameter model.  (Table 3). Interestingly, there were signi cant differences in the blood levels of white blood count, total bilirubin, ALT, ALKP and PLR ratio (   Validation of ndings using a non-transplant HCC dataset We evaluated a different dataset of small HCC patients who were not transplanted to con rm our ndings. Using the same 2-parameter model of blood lymphocyte counts and AST levels, we also showed a statistically signi cant difference between patients with small HCCs who had low 2-parameter values 2.6% of the small HCC patients), compared with patients with small HCCs having high 2-parameter values (97.4% of the small HCC patients), although with only 39 HCC patients (Table 5) . Abbreviations: Lymphs, lymphocytes (x10 9 /L); AST, aspartate amino transferase (IU/mL).

Discussion
This report shows that the combination of elevated blood levels of elevated lymphocyte counts and AST values are associated with presence of small size (≤3cm) HCCs, compared with non HCC chronic liver disease patients, both groups of whom were transplanted at our institute.
We validated the nding with a small set of non-transplant patients. Our plan is to test this 2-parameter combination on a future large HCC dataset, and then to consider evaluating the combination for use in the screening of chronic liver disease patients, who are thought to be at increased risk for subsequent HCC development.
The strength of this study is that these bloods parameters are routinely used blood tests in common clinical practice and are cheap. The weakness of the study is the fairly small HCC sample size of 101 HCC patients and the very small con rmation sample size. The reason for the latter is that adherence to screening guidelines is uncommon and most patients get diagnosed with quite advanced tumors, which were excluded from our study by design. Thus, we recognize that this is a preliminary nding and that con rmation from another, larger dataset is needed. If validated, this 2-parameter combination will need evaluation as a screening tool in a large group of prospectively followed patients with chronic liver disease, who have not yet developed HCC.
A likely reason these parameters might be useful is that HCC develops on the basis of a chronically in amed liver (7). Elevated lymphocytes represent a peripheral blood in ammation parameter, and AST elevation is a liver damage marker. In ammation has also been previously found to be more associated especially with smaller HCCs (8), as have other elevated liver function tests and white blood cell constituents (9). The nding of readily available and cheap biomarkers to better diagnose the presence of HCC at small and treatable size, is one of the main objectives of current HCC research.