Prognostic value of pretreatment serum alanine aminotransferase/aspartate aminotransferase (ALT/AST) ratio and gamma glutamyltransferase (GGT) in patients with esophageal squamous cell carcinoma

Esophageal cancer is one of the most prevalent malignant diseases worldwide, among all histologic types, esophageal squamous cell carcinoma (ESCC) occupies major portion [1, 2], and has the sixth mortality rates of any cancer globally. Most patients with ESCC will receive optional therapeutic options, such as surgery only, surgery with adjuvant chemotherapy, and adjuvant systemic therapy which may consist of radiotherapy, or a combination of these treatments. However, the overall 5-year survival rate of patients treated with only surgical resection is less than 20% [3, 4]. As treatment plans are becoming more individualized for each patient, it is important to assess disease progression in a timely manner while accurately evaluating the prognosis [5]. To date, various serum biomarkers, such as SCC, CYFRA21-1and CEA, have been served as the valuable markers to estimate the prognosis of ESCC patients [6]. However, the sensitivity and specificity are not sufficient or reliable. Furthermore, correlations between ESCC survival and thrombin time (TT) [7] or apolipoprotein A1 (Apo-A1) [8] have been reported in our previous study. Thus, in order to improve the posttreatment survival of patients, identification of more effective and accurate biomarker of ESCC is a necessity.

The routine blood sample that examines liver function tests (LFTs), partly consist of alanine aminotransferase (ALT), aspartate aminotransferase (AST), the level of ALT/AST ratio (LSR), total bile acid (TBA), gamma glutamyltransferase (GGT), and lactate dehydrogenase (LDH). LFTs are often included as routine tests for many different liver and non-liver diseases and are often obtained at initial consultation. Furthermore, changes in LFTs levels in cancer patients before and after neoadjuvant treatment are closely related to postoperative recurrence, such as breast cancer [9], gastric adenocarcinoma and other cancers. Serum ALT and AST are the circulating transaminases in the body, and are specific markers of liver dysfunction, which can generate products in gluconeogenesis and amino acid metabolism through catalyzing the transfer of amino groups [10, 11]. Many studies have indicated that serum levels of ALT and AST may be correlated with hepatitis tumors [12], type 2 diabetes mellitus [13], cardiovascular disease [14] and other diseases, and is the level of ALT/AST ratio (LSR). However, no studies were used to evaluate the relationship between the pretreatment serum LSR and survival of ESCC patients. Furthermore, as a key enzyme in glutathione (GSH) metabolism, GGT is the major antioxidant of the cell, which have played a key role in neutralizing reactive oxygen compounds and free radicals by catalyzing the degradation of extracellular GSH [15]. Previous studies have reported on the associations of serum GGT levels with the risk of cancer [16, 17]. This study was designed to conduct a retrospective cohort analysis to explore the predictive role of the LSR and GGT on overall survival (OS) in patients with ESCC.

In ESCC, prognostic biomarkers are needed to understand the development of cancer and for tailoring individual therapeutic strategies. Thus, it is imperative that inexpensive and convenient prognostic biomarkers for this disease are identified.

LFTs are routine laboratory tests, and earlier studies have noted the relationship of LSR, GGT and the risk of malignances, such as breast cancer [9], gastric cancer [20], liver cancer and other cancers. To date, the associations between LSR, GGT and ESCC survival have not been well developed. In our study, the cut-off points of LSR and GGT were defined as the median, which demonstrated that pretreatment serum LSR and GGT levels were related with 5-year OS in ESCC patients. Among the 447 ESCC cases examined in this retrospective study from Sun Yat-Sen University Cancer Center, we observed that patients with higher LSR levels showed significantly better prognosis compared with those patients with low LSR levels, not only in the entire cohort but also in the subgroups stratified by pathological stage (T1–T2 subgroup, T3–T4 subgroup, N0 subgroup and M0 subgroup). We also found that patients with a higher GGT showed significantly poorer prognosis than normal GGT patients, not only in the entire cohort but also in the subgroups classified by pathological stage (T3–T4 subgroup and N1-N2 subgroup). After adjustment for clinical characteristics, the elevated serum LSR and GGT were both associated with alcohol index and death.

The LSR and GGT tests are simple, inexpensive and widely used in clinical laboratories. As the major critical enzymes, ALT and AST generate products in gluconeogenesis and amino acid metabolism, and as specific markers of liver dysfunction, they catalyse the transfer of amino groups [10]. GGT is the major enzyme in the glutathione (GSH) catabolism, which also function as a biomarker for excessive alcohol intake [15]. Furthermore, many studies have demonstrated that LFTs have effects on the different hepatic injures, type 2 diabetes mellitus, cardiovascular disease and other diseases. In addition, several reports have confirmed the links between LFTs and prognosis in cancer patients. Chen el al. reported that the lower preoperative LSR level was found to be associated with decreased survival in patients with gastric adenocarcinoma [20]. Shen also suggested that pretreatment AST was related with the clinical outcome in hepatitis B-induced hepatocellular carcinoma after hepatectomy [21]. Preyer et al. identified that GGT was an independent risk factor in breast cancer over and above the alcohol consumption and other life style risk factors [16]. Mok et al. published a large study of new cancer cases, which occurred among 1,662,087 Koreans (ages 20–95 years, 1108,121 male and 553,966 female) who received health insurance from the National Health Insurance Service during 1995 and 1998. These patients were followed up for 17 years, and the elevated serum level of GGT was independently links with risk of various tumors, such as colorectal, stomach, lung and bile duct cancer [22]. However, the function of LSR and GGT in carcinogenesis is not well understood. It is interesting to consider the reason about the observed links between LSR, GGT levels and incident cancer risk, which may be attributed to its multiple properties, including anti-inflammatory and antioxidant properties. Moreover, serum LFTs are markers of alcohol intake, especially GGT. In our retrospective study, higher LSR and GGT levels were related to ESCC patients consumption of alcohol, and a significant relationship between increased GGT and alcohol intake has previously been reported [23, 24]. Numerous studies have indicated that alcohol consumption have influence on the risk of cancers of the oral cavity and pharynx, esophagus, stomach, liver and colorectum [25]. The possible mechanisms suggested include acetaldehyde being carcinogenic, mutagenic, influencing the ability to bind to DNA and protein, and destroying folate which results in secondary hyper-regeneration [26].

Cancer is a pro-inflammatory state, in which inflammatory cells actively participate in the occurrence of tumor development, such as tumor cell proliferation, survival, and migration [27‐29]. For LSR, the exact mechanisms underlying the association remain unclear: one explanation is that the change in LSR levels is associated with subclinical inflammation, which may result in continued damage of tissue precipitating some noninfectious diseases [30‐32]. The possible inference from LSR levels may be that they influence some of the pro-inflammatory mediators involved in carcinogenesis and affect tumor invasion and metastasis. Tumorigenic factors (inflammation and oxidative stress), which participate in these stages, have influenced on the cancer initiation in cascade of steps. The relationship between inflammation and cancer is very complex. Inflammatory factors were produced during the inflammatory response, including chemokines (e.g. CCL2, CXCL8) and cytokines (e.g. IL-6), which can recruit more inflammatory cells to the lesion site, such as neutrophilic granulocytes and mononuclear macrophages, and resulting in the inflammatory microenvironment [33, 34]. Moreover, as potent inflammatory mediators, TNF-α and IL-6 were released from stimulated T-cells and activated macrophages, which may result in continued damage to the liver tissue thereby influencing the LSR levels. [35]. The other possible explanation is that LSR levels may influence some of the pro-inflammatory mediators involved in carcinogenesis. Furthermore, through stimulating the growth of tumor cells and driving signaling transduction pathways, reactive oxygen factors leads to activation of redox-sensitive transcription species and genes which participated in the growth, proliferation, and survival of tumor cells.

Several potential mechanisms have been postulated for the relationship between GGT and cancer: 1. As the essential parts of the cellular defense apparatus, GGT and GSH have been used to against oxidative stress [15]. The elevated GGT levels in tumor cells can produce the reactive oxidant species (ROS), which may drive tumor progression; 2. Increased GGT has been regarded as a marker of exposure to certain carcinogens, which include persistent environmental pollutants including dioxins, lead, organochlorine pesticides and so on [36, 37]; 3. As a GGT isoenzyme, GGTII has been reported to a useful tumor marker in the diagnosis of small hepatocellular carcinoma [38]; 4. GGT levels can be affected by environmental and lifestyle factors (such as diet, smoking, and drinking) and genetic regulation [39]. However, further studied are needed to determine the underlying mechanisms.

Recently, serum markers have used in noninvasive cancer diagnosis, individual treatment, monitoring of prognosis and recurrence, and includes circulating tumor cells, exosomes, free DNA, and protein-based markers. As protein-based markers, the association between the LFTs and survival in ESCC were evaluated, and LSR and GGT were demonstrated to be independent predictors of overall survival in ESCC. Thus, LSR appears to be a new prognostic marker in ESCC.

In summary, this retrospective study is the first to validating the association between pretherapy serum LFTs and ESCC. Based on the 447 patients in our cohort, we showed that pretreatment serum LSR and GGT levels are prognostic factors for OS in ESCC, both in the entire cohort and in subgroups classified by pathological stage. These biomarkers offer high reproducibility and could be tested easily in clinical laboratories. As our study is a retrospective analysis, it is only valid for generating the hypothesis, a large-scale and prospective study should be done to validated the value of LSR and GGT in HCC.