Preoperative AminoIndex Cancer Screening (AICS) abnormalities predict postoperative recurrence in patients undergoing curative resection for non-small cell lung cancer

In total, 297 patients who underwent curative resection for NSCLC at the Osaka International Cancer Institute (Osaka, Japan) or Kochi University School of Medicine (Kochi, Japan) between November 2006 and September 2015 were enrolled. Patients were excluded if they had a history of other malignancies at the time of surgery for NSCLC. The clinicopathological characteristics of the enrolled patients are summarized in Table 1. Their mean age was 65.3 years (range, 30–86), and there were 187 males and 110 females. A total of 112 patients were non-smokers, while 185 were current or ex-smokers. Elevated preoperative serum CEA levels were observed in 72 patients. The surgical procedure selected was standard surgery (including lobectomy or more than lobectomy) for 224 patients and limited surgery (including segmentectomy or wedge resection) for 73, with all undergoing curative resection. The histological types were adenocarcinoma in 216 patients, squamous cell carcinoma in 61, and other types in 20. Regarding the pathological stage (p-stage), 163, 58, 9, 20, 30, 16, and one case were classified as stage IA, IB, IIA, IIB, IIIA, IIIB, and IV, respectively. Stage IV disease was due to a postoperative status of preceding brain metastasis. These stage classifications were based on the 7th Edition of the TNM classification [11].

Postoperative adjuvant therapy was administered to 61 patients: adjuvant chemotherapy to 58, adjuvant radiotherapy to 2, and adjuvant chemoradiation therapy to one. The regimen for adjuvant chemotherapy was as follows: oral UFT (Tegafur-Uracil) for 15 patients with stage I disease, for 2 with stage II disease, and for 1 with stage III disease. Gemcitabine (GEM) or vinorelbine (VNR) monochemotherapy was administered to one patient with stage I disease and 5 with stage II, respectively. Platinum (cisplatin or carboplatin)-based combined chemotherapy was administered to 4 patients with stage I disease, 6 with stage II disease, and 23 with stage III disease. Combined chemotherapy using GEM and VNR was administered to one patient with stage III disease. Most regimens were performed as described previously [12, 13]. Adjuvant radiotherapy was administered to two patients because of the short margin in the surgical stump of the bronchus in one and of the mediastinal field in the other. Adjuvant chemoradiation therapy was performed using the combined carboplatin-based regimen and 64 Gy on the surgical margin of the chest wall. No patients received preoperative therapy. Follow-up examinations were generally performed as follows: During the first 36 months after surgery, systemic and local screening examinations were conducted using blood tests, chest computed tomography (CT) scans were routinely obtained every 6 months, and fluoro-2-deoxyglucose positron emission tomography (FDG-PET) scans were performed where necessary. Brain CT or magnetic resonance imaging scans were also performed as required. During the first 2 years, these examinations were routinely performed and the findings obtained were carefully assessed. Thereafter, intensive examinations were performed as needed. Therefore, the median postoperative follow-up time was 6.0 years (range, 0.1 to 11.5 years) in this cohort.

The time of initial recurrence was assessed based on the onset of clinical symptoms, the detection of blood test abnormalities (e.g., serum CEA levels), or the detection of recurrent lesions on imaging, and the recurrence-free survival (RFS) period was defined as the period between surgery and the time of initial recurrence. When a tumor recurred, the initial recurrence site was evaluated and generally categorized into three patterns: local, distant, and multiple.

Patients underwent preoperative blood sampling within 1 week prior to surgery. Preoperative blood samples (5.0 mL) were collected from a forearm vein on the morning after overnight fasting using tubes containing ethylenediaminetetraacetic acid, and were immediately placed on ice. Plasma was prepared by centrifugation at 3000 rpm at 4 °C for 15 min and stored at − 80 °C until analyzed. Plasma samples were deproteinized using acetonitrile at a final concentration of 80.0%. PFAA concentrations were measured using high-performance liquid chromatography/electrospray ionization tandem mass spectrometry with pre-column derivatization, which have been described previously [8, 10, 14].

AICS (lung) values for patients with NSCLC were classified into the following three groups: rank A, B, or C. The AICS (lung) test detects lung cancer from plasma concentrations of serine, glutamine, alanine, histidine, ornithine, and lysine. AICS (lung) values range between 0.0 and 10.0, with 5.0 producing 80.0% specificity and 8.0 producing 95.0% specificity. A high AICS (lung) value was associated with a greater probability of lung cancer. Rank A was considered to be normal (0.0–4.9), rank B was relatively high (5.0–7.9), and rank C was high (8.0–10.0). Rank C indicated a high risk of lung cancer [1, 2, 8, 9].

RFS and overall survival (OS) curves were calculated using the Kaplan Meier method, and differences were examined by the Log-rank test. Cox’s proportional hazards regression model was used to perform a multivariate analysis of factors associated with RFS and OS. P-values were calculated using a one-way ANOVA for means or Fisher’s exact test for comparisons of rank. P < 0.05 was considered to indicate a significant difference. All statistical analyses were conducted using GraphPad Prism (software version 8; GraphPad Software Inc., San Diego, CA, USA) and R software (version 3.6.1; The R Foundation for Statistical Computing, Vienna, Austria).

This retrospective study was performed in accordance with the Declaration of Helsinki, and the study protocol was approved by the Ethics Committees of the Osaka International Cancer Institute (Osaka, Japan) (formerly the Osaka Medical Center for Cancer and Cardiovascular Diseases) (1404015008) and the Kochi University School of Medicine (Kochi, Japan) (ERB-000486). All participants gave their written informed consent for inclusion in this study. All data were analyzed anonymously.

The relationship between each rank and clinicopathological factors was summarized in Table 1. There were more elderly patients (p = 0.003) in rank C. Regarding the surgical mode, standard surgery (lobectomy or more) for lung cancer was more frequently selected for rank C (p = 0.053). No significant differences were observed in gender, the smoking status, preoperative CEA level, p-stage, or treatment method among patients with each rank.

The median follow-up period in the present study was 6.0 years, ranging between 0.1 and 11.5 years. There were 86 (30.0%) patients with tumor recurrence, and 42 (14.1%) died (Table 1). Tumor recurrence was observed in 22 (23.7%) patients with rank A, 15 with rank B (18.3%), and 49 with rank C (40.2%) (Table 1). The incidence of recurrence was significantly higher in rank C than in rank A or B (p = 0.0014). Based on this result, the following comparison was performed in two groups: rank A + B vs. rank C.

The 5-year RFS rate in all patients was 71.7%. RFS curves in ranks A + B and C were shown in Fig. 1. Five-year-RFS rates were 79.9% in rank A + B and 62.5% in rank C, and were significantly different (p = 0.000113). Figure 2 shows the RFS curves of these two groups according to the p-stage and histological type. A significant difference was observed in p-stage I (Fig. 1-a, p = 0.0026) and p-stage II (Fig. 2-b, p = 0.0402), but not in p-stage III (Fig. 2-c, p = 0.141). Regarding the histological type, only adenocarcinoma showed a significant difference in RFS (Fig. 2-d and 2-e, p = 0.0013). According to the univariate analysis of RFS in the present study, significant variables were the smoking status (non vs. current/ex), surgical mode (limited vs. standard), stage (I vs. II-IV), histological type (non-squamous cell carcinoma, non-Sq vs. squamous cell carcinoma, Sq), treatment method (surgery alone vs. surgery + adjuvant therapy), and preoperative AICS (lung) rank (rank A + B vs. rank C) (Table 2). Table 3 shows a multivariate analysis of RFS. Of the six variables tested, the p-stage, AICS (lung) rank, and treatment method were identified as significantly independent factors affecting RFS.

Forty-two (14.1%) patients died. Death was not associated with the AICS rank (Table 1). The 5-year OS rates were 89.5% in all patients, 91.5% in rank A + B, and 86.5% in rank C. A slight difference was observed in OS between rank A + B and rank C (Fig. 3, p = 0.0708).

Since a marginal difference was found in surgical mode among each rank (Table 1), the postoperative recurrence pattern was carefully examined. Table 4 shows the relationship between the AICS (lung) rank and initial recurrence site in this cohort. No significant differences were observed in the recurrence site between rank A + B and rank C.