Combined Detection of Free Fatty Acid (FFA) and High-Density Lipoprotein Cholesterol (HDL-C) is a Promising Pretreatment Biomarker for Predicting the Overall Prognosis (OS) of Neuroendocrine Tumours (NETs) in the Colorectum: A Case-Control Study.

Purpose The aim of the study was to evaluate the prognostic value of free fatty acid (FFA) and high-density lipoprotein cholesterol (HDL-C) in predicting colorectal neuroendocrine tumours (NETs). One hundred patients with pathologically diagnosed colorectal NETs in 2011-2017 were enrolled, and the levels of FFA, HDL-C, low-density lipoproteincholesterol (LDL-C), triglycerides (TGs), cholesterol (CHOL), apolipoprotein A1 (ApoA1) and apolipoprotein B (ApoB) between colorectal NET patients and healthy controls matched by age and sex were compared. In addition, the association of clinicopathological characteristics and follow-up data with FFA and HDL-C was analysed.


Introduction
Neuroendocrine tumours (NETs), previously described as "carcinoid tumours" in1907, are heterogeneous neoplasms arising in secretory cells of the diffuse neuroendocrine system (Yao et al. 2008). The Epidemiology and End Results (SEER) database indicates that the incidence of NETs has increased signi cantly, approximately 5 times, to 5.25/100.000 cases/year; of these, colorectal NETs account for approximately 49.6% of primary NET sites in the digestive tract and have an incidence and prevalence inferior only to those of colorectal adenocarcinoma (Frilling et al. 2012; Lawrence et al. 2011; Modlin et al. 2003). Although endoscopic mucosal resection, endoscopic submucosal dissection, surgery, radiation andchemotherapy have been used to treatlocalized and metastaticcolorectal NETs, the 5-year survival rate of NETs with lymph node metastasis (LNM) or distant metastasis remains disappointing, with veyear overall survival rates of approximately 54-74% and 28-44.1%, respectively (Bertani et al. 2018; Dasari et al. 2017).
Free fatty acids (FFAs) are triacylglycerol (TG) precursors that are needed to replenish TG stores in adipose, liver and muscle tissue through esteri cation; when hepatic glycogen is low and muscles need energy, the TGs in adipose tissue are broken down into FFAs for energy (Stich and Berlan 2004). FFAs are overexpressed in colorectal patients (Zhu et al. 2021), and abnormal fatty acid metabolismis associated with tumour cell metastasis in various cancers, including colorectal cancer (Kawaguchi et  However, evidence for the prognostic ability of FFAs and HDL-C and their relationship in colorectal NETs has never been reported. We designed this study to evaluate the prognostic ability of FFA and HDL-C in patients with colorectal NETs, compare FFA with HDL-C and analyse their combination.

Patients
Between 2011 and 2017, a total of 100 consecutive patients who received treatment for colorectal NETs in our hospital were enrolled. We constructed a database of retrospectively collected data from patient medical records, including clinical characteristics, pathological reports and survival during the follow-up period.
For local excision procedures, such as endoscopic submucosal dissection (ESD) and transanal excision (TAE), LNM was evaluated through computed tomography (CT) or magnetic resonance imaging (MRI) before the treatment and during the follow-up periods. The diagnosis of a metastatic lymph node was based on the following criteria: 1) Size criteria: short axis diameter greater than 8 mm for round lymph nodes and greater than 10 mm for ovoid lymph nodes; and 2) Morphological abnormalities: irregular contour and margin, unclear border, heterogeneous internal echoes or signal intensity. The tumour diameter refers to the longest diameter of the tumour according to pathology reports. For patients with distant metastases, tumour diameter was determined by endoscopic ndings before treatment. The baseline characteristics of patients with colorectal NETs are listed in Table 1. The healthy controls were matched by age and sex one by one to patients with colorectal NETs.

Pathological Diagnosis
Tumour

Inclusion Criteria
Patients who were treated in our centre for localized and metastatic colorectal NETs by pathological diagnosis from 2010 to 2017.  Figure 1.
The clinical and laboratory parameters, as well as the comparison between the high FFA and low FFA groups, are shown in Table 2; similar parameters and comparisons with regard to HDL-C are also shown.
Pearson's χ 2 test showed signi cant associations between FFA and lymph node metastasis (χ 2 = 5.964, P=0.015) and between HDL-C and tumour size (χ 2 = 5.647, P=0.017). However, no signi cant association was found between FFA and tumour size (P=0.142) or between HDL-C and lymph node metastasis (P=0.443). Neither FFA nor HDL-C was found to be signi cantly associated with sex (P FFA =0.542,  Univariable and multivariable analyses were performed to analyse the potential prognostic factors for survival. The median follow-up period of this cohort was 70 months (range: 1-130 months). Univariable analysis showed that higher serum FFA levels (≥0.52 mmol/l) (P=0.014), lower serum HDL-C levels (≤1.0 mmol/L) (P=0.022), the presence of tumour grades G2 (P=0.003) and G3 (P<0.001), positive lymph node metastasis (P<0.001), tumour size larger than 2 cm (P<0.001), and age older than 65 years (P=0.046)were signi cantly associated with shorter OS. Multivariable analysis showed that tumour grade G3 and positive lymph node metastasis in the FFA group (P G3 =0.013, P LNM =0.048) and HDL-C group (P G3 =0.017, P LNM =0.016) were signi cantly associated with shorter OS. There was no signi cant association between shorter OS and tumour size/FFA/HDL-C. The hazard ratio (HRs) and corresponding 95% con dence intervals are shown in Table 3.  Table 4.  Reduced plasma levels of HDL-C are a hallmark of obesity and cardiovascular diseases (CVDs); similarly, reduced ApoA1has also been associated with cardiovascular risk (Raitakari et al. 2013; Su and Peng 2020). Due to component differences between ApoA1 and HDL-C, a similar association was not found for LNM, tumour size or poor survival with HDL-C. However, the level of ApoA1 in colorectal NETs was signi cantly lower than that in controls, and the area under the receiver operating characteristic (ROC) curve was 82.2%. Interestingly, the potential diagnostic ability is worth analysing.
There were some limitations in our study. First, it was of a retrospective design and included a relatively small number of patients. However, we believe the results are reliable. Because this study lasted more than 130months, we could investigate the long-term survival outcomes and prognostic factors after different treatments, even with the small number of patients. Second, progression-free survival (PFS) data were not collected, and prognostic results could not be predicted comprehensively. Finally, further studies should be performed to validate our main conclusions.

Conclusions
FFA, CHOL, and ApoB were overexpressed in colorectal NETs, and HDL-C and ApoA1 were underexpressed. FFA ≥ 0.52 mml/L can be considered a cut-off point to predict LNM and poor OS, HDL-C≤1.0 mmol/L can be considered another cut-off point to predict tumour size ≥2 cm and poor OS, and the combined detection of FFA and HDL-C complementarily predicts OS.   Kaplan-Meier survival curves for colorectal NET patients with different tumour grades, tumour sizes and lymph node metastases, as well as with respect to pretreatment FFA and HDL-C levels in serum.