Patients and tissue samples
Retrospective frozen tissue samples of primary lung adenocarcinoma obtained from patients who underwent radical surgery from January 2013 to December 2016 at Hamamatsu University Hospital were examined. Radical surgery was defined as complete resection performed with lobectomy or pneumonectomy accompanied by systematic lymph node dissection at stage I or II, and as complete resection achieved by segmentectomy or wedge resection with or without lymph node sampling at stage I. Tissue samples of primary tumors were collected immediately after the resection and stored at −80°C after a rapid freezing in liquid nitrogen. Histopathological diagnosis was performed by experienced pathologists according to the World Health Organization criteria. Pathological staging was identified based on the 8th edition of the TNM classification for lung and pleural tumors (22). Patients were followed-up with computed tomography (CT) of the body trunk and biochemical examination of carcinoembryonic antigen (CEA) every 3 months during the first 2 years, then, every 6 months until more than 5 years after the surgery. When CEA was elevated (≥5.0 ng/mL) without any CT findings of recurrence, head magnetic resonance imaging, and systemic positron emission tomography were performed for the detection of brain metastasis or bone metastasis.
In patient selection, clinical records of these tissue samples were retrospectively reviewed. Patients with pathological stage I or II indicated for radical surgery and with major histological subtypes of invasive adenocarcinoma (lepidic, papillary, acinar or solid predominant) were analyzed. Patients who received induction chemotherapy or radiotherapy and those with other subtypes of adenocarcinoma were excluded.
Then, cases without and with recurrence were assigned to non-recurrent and recurrent groups, respectively. Recurrence was defined as radiological imaging-based findings of distant or locoregional recurrence within 5 years, whereas no recurrence was defined as no findings of distant or locoregional recurrence in ≥5 years after the radical surgery. In the non-recurrent group, cases with follow-up period of <5 years were excluded. In the recurrent group, cases with recurrence in the form of pleural dissemination were excluded, assuming the possible attribution with insufficient surgical margin. Finally, 10 cases for recurrent and 10 for non-recurrent groups were subjected for analysis.
Histological evaluation
Paraffin-embedded tissue blocks were sectioned at 3 μm thick. Sections stained by hematoxylin–eosin (HE) were examined for adenocarcinoma subtype, tumor size, lymph node metastasis, and STAS. D2-40 stain was used to evaluate lymphatic vessel invasion and Elastica van Gienson stain to evaluate blood vessel invasion. All histological sections were reviewed by experienced pathologists.
Chemicals
Methanol, chloroform, glacial acetate, and ultrapure water were purchased from Wako Pure Chemical Industries (Osaka, Japan). The 1,2-dilauroyl-sn-glycero-3-PC (Avanti Polar Lipids, Alabaster, AL), PC(12:0_12:0), was used to calibrate standard lipid levels.
Lipid extraction from the cancer tissue
Each weight of the frozen tissue samples was measured using Sartorius analytical lab balance CPA224S (Sartorius AG, Göttingen, Germany) (Additional file 1, Supplemental Table). After the weight measurement, Modified Bligh-Dyer methods were performed for lipid extraction. Tissue samples were transferred into glass tubes, and 0.34 ml of methanol, 0.17 ml of chloroform, and 0.14 ml of 0.322 M glacial acetate were subsequently added. Then, 1.6 mmol of PC(12:0_12:0) per 1 mg of sample tissue was added and subsequently followed by 10-min extraction at room temperature. After the extraction, 0.17 ml of chloroform was added and vortexed, sequentially, 0.17 ml of 0.322 M glacial acetate was added and vortexed. Extracted samples were subjected to centrifugation at 3,000 rpm for 10 min. Extracted organic layers were transferred into new glass tubes and were evaporated until completely dried using miVac Duo LV (Genevac, Ipswich, England). The extracted lipid was dissolved with 20 μl of methanol, and 2 μl of the dissolved lipids were diluted again with methanol proportional to the weight of the original tissue samples so that the concentration of PC(12:0_12:0), internal control, will be as similar as possible among cases.
Lipid analysis by liquid chromatography–tandem mass spectrometry (LC–MS/MS)
Extracted lipids from collected frozen tissue samples were analyzed using Q Exactive™ Hybrid Quadrupole-Orbitrap™ Mass Spectrometer equipped with an electrospray ionization source and connected to an Ultimate 3000 system (Thermo Scientific). 10 μL of the extracted lipid samples were injected and separated on Acculaim 120 C18 column (150 mm × 2.1 mm, 3 μm) (Thermo Scientific). Components of mobile phase A were as follows: water-acetonitrile-methanol (2:1:1 v/v/v), 5 mM ammonium formate, and 0.1% formic acid. The components of mobile phase B were as follows: acetonitrile-isopropanol (1:9 v/v), 5 mM ammonium formate, and 0.1% formic acid. For elution, the flow rate was set at 300 μL/min. A set of linear gradient starting at 20% solvent B was used and linearly increased to 100% B in 50 min, maintained at 100% B until 60 min, then decreased linearly to 20% B from 60 min to 60.1 min, and finished with 20% B for the last 10 min. The overall run time was 70 min. MS instrument conditions were as follows: sheath gas flow rate, 50; auxiliary flow rate, 15; sweep gas flow rate, 0; capillary temperature, 250°C; S-lens RF level, 50; probe heater temperature, 350°C; and spray voltage of 3.5 kV in positive mode and 2.5 kV in negative mode. Full-MS mode conditions for quantification were as follows: MS scan range, 220–2000; resolution, 70,000; AGC target, 1 × 106 and maximum injection time was 100 ms. For identification, top 5 data-dependent MS2 method with a resolution of 17,500 was used. The AGC target was 1 × 105, and the maximum injection time was 80 ms. Stepped normalized collision energies of 25.5, 30, and 34.5 for the positive mode and 19.5, 30, and 40.5 for the negative mode were applied. Spectral data were acquired in the m/z range of 220–2000 m/z using an Xcalibur v3.0 Software (Thermo Scientific).
Lipid identification and quantification
LipidSearch™ software version 4.2.13 (Mitsui Knowledge Industry, Tokyo, Japan) was used to identify and quantify lipid species. Parameter settings for identification were followings: database, HCD; retention time, 0.01 min; search type, product_QEX; precursor tolerance, 5.0 ppm; and product tolerance, 8.0 ppm. Identification quality filters of A, B, and C were used. Quantification was performed at m/z tolerance of ±0.01 with retention time range from −1.0 min to 2.0 min. Alignment of the identified lipid species among 20 cases was performed with retention time tolerance of 0.25. Molecules that are annotated as redundant lipid names with different calculated m/z and retention times were regarded as independent isomers (annotated as “Duplication” in Additional file 2).
Data processing
Trend analysis between the non-recurrent and recurrent groups was performed by comparing the average total lipid level between the two groups and principal component analysis (PCA). Intensities of lipids recorded in the Xcalibur v3.0 software and monoisotopic peak area values of lipid species identified by LipidSearch™ software were normalized by dividing with the area values of internal control, PC(12:0_12:0). The total lipid level of each case was defined as an accumulation of normalized intensities of lipids. Normalized area values were subjected for PCA.
For respective lipid species, P-values were calculated using the Student t-test to compare area values between the two groups. To screen candidate lipids for recurrence prediction, lipidomes were compared between the non-recurrent and recurrent groups by describing volcano plots with -log10 (P-value) for vertical axis and log2 (folding change) for horizontal axis. The folding change for a lipid was defined as an average area value of the recurrent group divided by that of the non-recurrent group. Significance was determined at P-values of <0.05, folding change of ≥2.0 or ≤0.5.
Statistical analysis
Demographic information and associations with clinical characteristics were evaluated using the Fisher exact test (categorical variables) or the Mann–Whitney U-test (for continuous variables). The Student t-test was used to compare the average total lipid amounts of the non-recurrent and recurrent groups and to describe volcano plots. Recurrent-free survival (RFS) was determined as the time from operation until the first disease recurrence or death. Survival curve was described using the Kaplan–Meier method. The optimal cut-off values to discriminate the two groups were determined using the receiver operating characteristic (ROC) curve analysis. The area under the ROC curves (AUCs) were calculated to validate the discrimination abilities of candidate lipids. Spearman’s rank correlation analysis was used to validate the correlation among candidate lipid predictors. All statistical analyses except for the t-test were performed using R (The R Foundation for Statistical Computing, Vienna, Austria, version 3.6.2). The Student t-test was performed with “TTEST” of Excel™ (Microsoft, Redmond, USA). P-values of <0.05 were considered as significant.