Background
Colorectal cancer (CRC) is the third most commonly diagnosed cancer and the fourth leading cause of cancer-related death in the world. By 2030, its burden is expected to increase by 60% to more than 2.2 million new cases, with 1.1 million deaths [
1,
2]. In Japan, CRC has a great societal impact [
3].
The progression of CRC is generally slow, and its symptoms are not readily apparent, with almost no subjective symptoms in the early stages. Surgical resection is an effective treatment for CRC, and radical resection of localized tumors achieves high survival rates [
4,
5]. However, the 5-year survival rate at stage IV is extremely poor (~ 18.8%) [
6]. Moreover, CRCs recur after surgical resection in ~ 10% of patients with stage II disease and in ~ 20–40% with stage IIIa–IIIb disease; hence, recurrence remains an important concern [
7,
8].
Recent advances in analytical techniques [e.g., liquid chromatography mass spectrometry LC/MS)] and metabolomics analysis (e.g., in vivo amino acid profiling) have been reported in the context of various diseases, including cancer. Plasma-free amino acid (PFAA) profiles have been shown to differ in healthy individuals and patients with cancer or other diseases, owing to complex metabolic changes [
9,
10]. Based on our previously described “AminoIndex technology”, which assesses changes in PFAA levels via multivariate analysis [
11,
12], we developed a cancer screening method termed “AminoIndex Cancer Screening (AICS)” [
13,
14]. AICS is a novel means of evaluating the probability of cancer, and we are currently using it in clinical practice to screen for multiple cancer types.
AICS consists of a combination of 6 amino acids that are differentially expressed in different cancer types. It discriminates between changes in the levels of amino acids that are characteristic of a specific cancer type and those that are common among cancer types [
13,
14], and statistically analyzes the differences. Based on the amino acid data, it evaluates the probability of the individual currently having cancer. Probability is ranked on a scale of 0.0–10.0, in which a higher number represents a higher probability. Risk is ranked as A, B, or C, with rank C representing the highest risk [
14].
Although tumor markers such as carbohydrate antigen 19–9 (CA19–9) and carcinoembryonic antigen (CEA) can be easily quantified in blood tests, their levels may not be increased in early-stage cancers [
15]. AICS detects cancer-related changes in amino acid profiles even in patients with stage I cancers, and the profiles do not change as the cancer progresses [
14]. Moreover, because AICS is a simple examination requiring only blood sampling, it can be performed during regular health check-ups.
Although assessment of the clinical usefulness of AICS is ongoing, it remains unclear why the PFAA levels fluctuate (e.g., in response to tumor-bearing status) and whether such fluctuations trigger cancer. Further elucidation of the biological mechanisms underlying the changes in the PFAA levels might allow for the development of both static and dynamic models of carcinogenesis via system analysis. System analysis of cancer patients based on systemic amino acid metabolism provides information on the nature of the cancer and, by doing so, may not only facilitate early detection but also the formulation of treatment, prognostic, and relapse monitoring strategies.
In this preliminary study, the preoperative and postoperative PFAA profiles, which are indicative of tumor-bearing status, were compared in patients with CRC via AICS.
Discussion
The amino acids in the plasma are maintained at constant levels by homeostatic processes in the body. Metabolomics analysis (e.g., in vivo amino acid profiling) of various disease states has shown alterations in the PFAA profiles owing to collapsed regulatory mechanisms; these disease states include cancer, liver failure, kidney failure, Alzheimer’s disease, and psychiatric disorders. Via metabolomics analysis, AICS statistically compares PFAA profiles between patients with CRC and healthy individuals; consequently, it can determine whether an individual has CRC. Although clinical application of this screening method has widened recently, the biological mechanisms that control the PFAA levels remain unknown, as does the cause and effect relationship between the PFAA levels and cancer. Hence, whether fluctuations in the PFAA levels cause cancer or vice versa has yet to be determined.
The present study showed a significant decline in the AICS values after surgical resection in patients with primary CRC and a preoperative rank of B + C. The decline was stage-independent, even occurring in patients with right-sided tumors or poorly differentiated adenocarcinomas, both of which are highly malignant (100% reduction in the AICS value and rank in both conditions). These results indicate that elimination of cancer cells restores the PFAA levels to precancer levels, as determined in vivo. Previous animal experiments have suggested that release of the nuclear protein HMGB1 into the blood, which affects the metabolism of distant organs, accounts in part for the altered PFAA levels in cancer patients [
21]. Alterations may also result from interactions between cancer cells, with involvement of the immune system [
22]. However, assessment of postoperative changes using the mGPS, a prognostic indicator, showed no significant difference in this study, although the proportion of patients classified as group I tended to increase.
The results of this study suggest that changes in the PFAA levels in patients with CRC strongly reflect CRC-bearing conditions; i.e., the cancer causes the changes in the PFAA levels. This point should be further clarified, as recurrence of CRC after resection is an important problem.
Tumor markers are often used to monitor for relapse after CRC resection. However, in the present study, the sensitivities of CEA and CA19–9 for detecting CRC were low, similar to in a previous study of early-stage CRC [
15]. Moreover, these markers were significantly less sensitive than AICS for the detection of CRC [
15].
Although previous studies have compared the preoperative and postoperative PFAA profiles in patients with breast, stomach, and thyroid cancers [
23,
24], there have been no comparable investigations in patients with CRC. We previously observed changes in the levels of 18 different amino acids after treatment of various cancers; however, the interpretation of the results was cumbersome, because some amino acids increased in abundance whereas others decreased [
23]. In the present study, the use of only 6 amino acids (identified via the AminoIndex technology using multivariate analysis scores) for determination of AICS values simplified interpretation of the data and provided information regarding the probability of cancer.
Recent reports have indicated that right-sided CRCs are more malignant than left-sided CRCs and have a worse prognosis [
25]. The complexity of the colorectal region and the different characteristics of left-sided versus right-sided CRCs have stimulated discussions about the selection of therapeutic agents [
26]. In 2016, the American Society of Clinical Oncology reported that many of the variables associated with right-sided tumors were major indicators of poor prognosis [
27,
28]. According to previous reports, 20–30% of CRCs occur on the right side and 70–80% on the left side [
6]. Similarly, in our study, about 60% of CRCs with a rank of B or C were on the left side [
29]. The results of our study show that resection of highly malignant right-sided tumors significantly reduces the AICS value. This finding suggests that assessment of treatment efficacy is necessary at earlier stages for right-sided CRCs compared with left-sided CRCs.
Our study examined changes in the preoperative and postoperative PFAA profiles in patients with CRC via AICS, which is a cancer probability assessment test. We suggest that cancer cells alter the PFAA profiles, which is reflected in the AICS value. This premise has important clinical implications but requires verification.
Several aspects of our study require discussion and further testing. First, in 5 patients, the AICS value did not decline after resection. All 5 patients were classified with left-sided CRC, and postoperative adjuvant chemotherapy was administered to 4 of these cases. The four cases of moderately differentiated cancers demonstrated a deeper invasion depth than the one case with highly differentiated cancer; many cases have been previously reported showing vascular invasion, lymph node metastasis, and peritoneal dissemination [
30]. In regard to the one case of highly differentiated type, it is necessary to collect and verify similar cases in the future. Although combining surgery and chemotherapy is effective for these high-risk cases, the efficacy of postoperative adjuvant chemotherapy for stage II CRC has not yet been established in Japan. Moreover, implementation of adjuvant chemotherapy largely depends on hospital policy and thus currently differs among hospitals [
4]. Two of the above 5 patients were confirmed to have recurrence postoperative (Additional file
3). However, for the remaining 3 cases, it remains unknown why the AICS value did not decrease.
Second, the present study excluded subjects classified as AICS rank A. These patients showed no postoperative changes in their PFAA profiles, which were within the AICS tolerance range for sensitivity, specificity, and positive predictive value (Additional file
4). However, investigations that consider factors such as patient background, history of other diseases, and drug compliance status are needed.
Third, the timing of postoperative follow-up blood collection should be considered. Because this study was an exploratory study, blood was collected 6 or more months after resection, but the range varied widely, from 0.5–6.5 years (median, 4.1 years). However, there was no significant correlation between the AICS value and the blood collection time (Additional file
2). Furthermore, the relationship between amino acid metabolism, the PFAA profile, and the amount of time between surgical trauma and wound healing is unknown [
22]. In this study, only 5 cases showed recurrence during the follow-up. In the future, the number of cases will be increased prospective verification with predetermined blood collection time points and long-term studies with follow-up until recurrence are needed.
This study showed that the PFAA profile reflects the tumor-bearing status in patients with CRC. AICS might be an effective way to predict prognosis and monitor recurrence and the patient’s clinical course postoperatively.
In the future, it will be essential to clarify through ongoing research whether elimination of factors indicative of poor prognosis affects the degree of AICS value reductions.