From a follow-up period of up to 130 months, we collected consecutive colorectal NET patients by pathological diagnosis from 2011-2017 and found that FFA ≥ 0.52 mml/L can be considered a cut-off point for predicting LNM and poor OS, HDL-C≤1.0 mmol/L can be considered another cut-off point for predicting tumour size ≥2 cm and poor OS, and FFA/HDL-C >0.75 predicted tumour grade, LNM, tumour size and even worser OS in our study.
According to the updated guidelines for colorectal NETs in 2016[7], tumour grade, size, symptoms, and treatment modality were only significant in univariate analysis, and stage was the strongest predictor of survival in multivariate analysis; on the one hand, tumour size and depth can predict LNM; on the other hand, size remains a less than totally reliable discriminator of prognosis, and factors predicting LNM remain more researched. Currently, whether serological indicators can predict prognosis in colorectal NETs has not been deeply demonstrated. In our study, we identified a new biomarker of FFA/HDL-C that has a promising ability to predict prognosis as well as tumour grade, size and LNM. In addition, the predictions of FFA, HDL-C and FFA/HDL-C were relatively independent and complementary, and the OS prognosis of FFA/HDL-C was even worse than that of FFA or HDL-C. Combined detection of FFA and HDL-C is a promising biomarker in colorectal NETs, and it may benefit new guidelines in the future.
In clinical work, we found that in some cancer patients, traditional tumour markers such as carcinoembryonic antigen (CEA) and carbohydrate antigen 19-9 (CA19-9) were elevated tens of times compared with the upper limit of the healthy reference, while the standard deviations of lipid metabolism biomarkers in patients and controls were relatively similar. In our study, we found that the FFA/HDL-C ratio of cancer patients has more scatter points than other lipid metabolism biomarkers, as shown in Figure 1. These scatter points, which have similar changes in FFA/HDL-C to CEA and CA19-9, account for almost one tenth of the scatter points. Similarly, we found that one-tenth of patients whose FFA/HDL-C >0.75 had a worse prognosis than the others, as shown in Figure 2. Interestingly, these scatter points may play an important role in predicting tumour grade, size, LNM and poor prognosis.
Although the patient numbers of FFAs ≥0.52 mmol/L and HDL-C ≤1.0 mmol/L were low, our results and their combined detection were credible. Firstly, the Surveillance, Epidemiology and End Results (SEER) database indicates that the incidence of NETs has increased significantly, 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[2]. Secondly, it is worth noting that patients who had FFA ≥0.52 mmol/L accounted for almost half of the total colorectal patients, and those with HDL-C ≤1.0 mmol/L accounted for almost one-fifth of the total; interestingly, patients with FFA ≥0.52 mmol/L and HDL-C ≤1.0 mmol/L also made up one-fifth of the patients with FFA ≥0.52 mmol/L.
Traditionally, the level of FFAs has been detected to assess lipid metabolism and has been associated with hypertension, cardiovascular disease, type 2 diabetes, and obesity[17].Similarly, HDL-C, LDL-C, TG, CHOL, ApoA1 and ApoB, all widely used, were analysed to assess lipid metabolism in the body as combined biochemical indicators. Recently, an increasing number of researchers have indicated that FFAs and HDL-C are involved in colorectal cancer. FFAs are overexpressed in colorectal cancer [9], dietary palmitic acid promotes cancer metastasis [18], and the reprogramming of fatty acid metabolism plays an important role in LNM of various cancers by the fatty acid-binding protein 5 (FABP5) pathway [10, 11].Interestingly, LNM and poor prognosis were significantly associated with the pre-treatment FFA level of colorectal NETs, consistent with our results. In addition, oxidative modification of HDL results in compositional and functional changes, and following increased cholesterol ester transfer protein (CETP) activity in parallel with decreased lecithin–cholesterol acyltransferase (LCAT) activity, HDL particles become larger, and changes in HDL composition, such as enrichment with TG and reduced ApoA1, paraoxonase-1 (PON1) and apoM and increased serum amyloid A (SAA) proteins, occur. The interaction of overexpressed SAA with TLR2 in cancer cells leads to cancer progression throughthe NF-κB-mediated pathway [19]. In addition, HDL-C was related to poor prognosis in patients with colorectal cancer, and very low levels of HDL-C (<30 mg/dL) in women were significantly associated with cancer mortality[13, 14]. In our study, tumour size (≥2 cm) was related to pre-treatment HDL-C level, and worse OS was found in colorectal NETs with larger tumour size, consistent with the above researchers.
CgA (chromogranin A), synaptophysin (Syn) and CD56 are three neuroendocrine differentiation (NED) immunohistochemistry markers frequently used in NETs [20–22]. The results of immunohistochemistry are usually marked by semiquantitative scores to show positive cell percentages and positive cell staining intensities but are limited to qualification and by pathologist experience. Reports of CgA, Syn and CD56 are difficult to standardize, and it is difficult to predict prognosis by immunohistochemistry results directly. CgA in serum is an important biomarker in advanced pancreatic cancer and metastatic neuroendocrine tumours[23, 24], but due to the lower incidence of NETs[25] and the high cost of detecting reagents, the serological CgA test has not been widely performed. FFA and HDL-C are common biochemical biomarkers detected in clinical laboratories, and they have promising applications in predicting LNM and tumour size and predicting the OS of colorectal NETs.
The median survival and 5-year survival of patients also consistent with our conclusion. The median survival of patients with FFA ≥0.52 mmol/L was 67.8±7.3 months, the median survival of those with HDL-C ≤1.0 mmol/L was 53.8±9.7 months, and the median survival of those with FFA ≥0.52 mmol/L and HDL-C ≤1.0 mmol/L was only 45.9±14.3 months. The 5-year survival of patients with FFA ≥0.52 mmol/L was 50.9%, that of patients with HDL-C ≤1.0 mmol/L was 47.4% and that of patients with FFA ≥0.52 mmol/L in combination with HDL-C ≤1.0 mmol/L was only 40.0%. Therefore, patients with high FFA and low HDL-C had a worse prognosis than patients with only high FFA, only low HDL-C and patients with low FFA or high HDL-C. FFA/HDL-C predicted an even worse prognosis in terms of OS.
Mixed adenoneuroendocrine carcinoma of the colon and rectum are rare cancers; they are characterized by the presence of a combination of epithelial and neuroendocrine elements, where each component represents at least 30% of the tumour[26] and are unmet areas where NETs need to be described and defined[27]. In our study, there were more colorectal NET patents with positive LNM than with negative LNM, which may be due to not excluding mixed adenoneuroendocrine carcinoma among the patient groups, which is composed of poorly differentiated neuroendocrine carcinoma and easily metastasizes[26, 28].
Reduced plasma levels of HDL-C are a hallmark of obesity and cardiovascular diseases (CVDs); similarly, reduced ApoA1 has also been associated with cardiovascular risk[29]. 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 significantly 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. In addition, no significant association with pathological characteristics was found in CHOL and ApoB in our study.
There were some limitations in our study. First, it was of a retrospective design and included are relatively small number of patients. However, we believe the results are reliable. Because this study lasted more than 130 months, 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.