Patients and samples
A total of 599 consecutive primary invasive breast cancer patients with available tissue specimens collected during surgery between 1992 and 2008 at the Department of Breast Surgery, Nagoya City University Hospital, Japan, were included in the study. All 599 tissue specimens were measured for TINAGL1 mRNA expression. Of these patients, 299 patients from 2000 to 2008 had invasive breast cancer tissue specimens available as tissue microarrays (TMAs), and these specimens were used to evaluate TINAGL1 protein expression. The tissue specimens obtained during surgery were snap-frozen in liquid nitrogen immediately after resection and stored at −80°C until RNA extraction or fixed in 10% buffered formalin and embedded in paraffin. Histological grade was estimated using the Bloom and Richardson method, as proposed by Elston and Ellis (1991). Adjuvant therapy was conducted at the physician’s discretion according to each patient’s clinicopathological features. Patients who underwent neoadjuvant chemotherapy were excluded from the study to eliminate any biological effects of neoadjuvant chemotherapy on the tumor tissues. Patients who underwent adjuvant trastuzumab therapy were also excluded to avoid mixing patients who did and did not receive adjuvant trastuzumab treatment for HER2-positive breast cancer. Disease-free survival (DFS) was defined as the interval from the date of primary surgery to the earliest occurrence of one of the following: locoregional recurrence, distant metastasis, or death from any cause. Overall survival (OS) was defined as the interval from the date of primary surgery to death from any cause. Written informed consent forms for comprehensive research use were obtained from all patients before surgery. The study protocol was approved by the Institutional Review Board of Nagoya City University Graduate School of Medical Sciences (approval number: 70-00-0166) and conformed to the guidelines of the Declaration of Helsinki.
RNA extraction and reverse transcription-quantitative polymerase chain reaction (RT-qPCR)
Total RNA was extracted from the frozen breast cancer tissue specimens using an RNeasy Mini Kit (Qiagen, Tokyo, Japan) according to the manufacturer’s protocol. The quantity of total RNA was measured using a DS-11 Spectrophotometer (DeNovix, Wilmington, DE, USA). cDNA was reverse-transcribed from the total RNA samples using a High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s protocol. TaqMan Gene Expression assays (Thermo Fisher Scientific) were used to measure the mRNA expression levels of TINAGL1, SEC23A, and the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Duplex RT-qPCR assays were performed using a TaqMan StepOnePlus Real-time PCR System (Thermo Fisher Scientific). The reactions was analyzed using a FAM-labeled probe for TINAGL1 or SEC23A (Thermo Fisher Scientific) and a VIC-labeled probe for GAPDH (Thermo Fisher Scientific) as a single assay for each sample. The amplification reaction mixture comprised 2 μL of cDNA, 10 μL of TaqMan Fast Advanced Master Mix (Thermo Fisher Scientific), 1 μL of TaqMan FAM-labeled probe, 1 µL of TaqMan VIC-labeled probe, and 6 μL of DEPC-treated water (Thermo Fisher Scientific) in a total volume of 20 μL. The RT-qPCR conditions were 95°C for 20 s, followed by 40 cycles of denaturation at 95°C for 1 s and annealing and extension at 60°C for 20 s. The results were converted into relative concentrations using a standard curve. The mRNA expression levels of TINAGL1 or SEC23A were normalized by the expression level of the housekeeping gene GAPDH as an internal control. We determined the cut-off levels for TINAGL1 and SEC23A mRNA expression as the median expression.
The Thermo Fisher Scientific assay numbers for TINAGL1, SEC23A, and GAPDH were Hs01115879_m1, Hs00197232_m1, and 99999905_m1, respectively.
Immunohistochemistry (IHC) of ERα, PgR, HER2, and Tinagl1
A single 4-μm-thick section from each paraffin-embedded specimen was initially stained with hematoxylin and eosin to verify that invasive carcinoma cells were present in sufficient numbers and that the quality of fixation was adequate for IHC analysis. Subsequently, 4-μm-thick serial sections were prepared from suitable tissue blocks and float-mounted on adhesive-coated glass slides for immunostaining of ERα (Dako Envision FLEX-ER; EP1; Agilent Technologies, Santa Clara, CA, USA), PgR (Dako Envision FLEX-ER; PgR636; Agilent Technologies), and HER2 (HercepTest II; Agilent Technologies) using an Autostainer Link 48 (Agilent Technologies). The immunostained specimens were scored after the entire sections had been evaluated by light microscopy (BX51; OLYMPUS, Tokyo, Japan).
Expression of ERα or PgR was evaluated by the proportion and intensity of positively-stained tumor cells using Allred’s procedure, with positivity defined as ≥1% of tumor cells exhibiting staining in the nucleus (Harvey et al. 1999). ERα-positive and/or PgR-positive specimens were considered hormone receptor-positive. HER2 expression was evaluated by the membrane staining pattern and scored on a scale of 0 to 3+ (Hicks et al. 2005). Tumors with a score of 3+ were considered HER2-positive. Tumors with a score of 2+ were tested for gene amplification by fluorescence in situ hybridization (FISH) using the PathVysion assay (Abbott Laboratories, Abbott Park, IL, USA) according to the manufacturer’s protocol. A ratio of >2.0 for HER2 gene/chromosome 17 was considered HER2-positive. Scores of 0, 1+, or 2+/FISH-negative were considered HER2-negative.
For IHC analysis of TINAGL1, TMAs of 2-mm diameter were created on slides after confirming that a sufficient number of invasive carcinoma cells were present and that the quality of fixation was suitable for IHC analysis. The primary antibody was a rabbit polyclonal anti-TINAGL1 antibody (Atlas Antibodies, Stockholm, Sweden) used at 1:30 dilution. Immunostaining was performed using a Leica Bond-Max Automated System and a Leica Refine Detection Kit (Leica Biosystems, Wetzlar, Germany). The TMA slides were scanned at 20× using Aperio Scanscope CS2 (Leica Biosystems). To ensure objectivity, the TINAGL1 protein expression level in the cytoplasm was evaluated by quantifying the staining intensity according to the H-score using a digital pathology system (eSlide Manager Application; Leica Biosystems) The H-score produced by this digital image analysis tool was previously shown to overcome the limitations of visual quantitative scoring by enabling objective and highly reproducible quantification of biomarkers (Bankhead et al. 2018; Ram et al. 2021). The H-score was calculated as follows: classification of cytoplasmic staining intensity into three categories (1, weak; 2, moderate; 3, strong), followed by multiplication of the percentage of stained cells by each category number, and finally addition of the values. In short, the H-score was calculated using the following formula: 1×(% cells in weak category) + 2×(% cells in moderate category) + 3×(% cells in strong category). For survival analysis based on TINAGL1 protein expression, the median H-score was set as the cut-off level. The Atlas Antibodies assay number for Tinagl1 was HPA048695.
We also evaluated TINAGL1 protein expression using the staining index (SI) method reported by Shen et al. (2019), which incorporates the intensity and percentage of positively-stained tumor cells. The intensity of the staining was scored as follows: 0, no staining; 1, weak; 2, moderate; 3, strong. The percentage of stained tumor cells was scored as follows: 0, 0%; 1, <10%; 2, 10%–50%; 3, >50%. SI scores >4 were defined as high TINAGL1 expression (Shen et al. 2019).
Statistical analysis
The associations of TINAGL1 mRNA expression level with clinicopathological factors were assessed by Student’s t-test and Fisher’s exact probability test. The Mann–Whitney U test was used to compare differences in TINAGL1 or SEC23A mRNA expression levels between two groups, and comparisons between three groups were adjusted by the Bonferroni method. Spearman’s rank correlation coefficient was used to evaluate correlations between two factors, such as TINAGL1 mRNA and TINAGL1 protein expression levels. Survival analyses were performed using the Kaplan–Meier method and verified by the log-rank test. DFS was censored on the last follow-up date if patients were relapse-free and alive, and OS was censored on the date when patients were last confirmed alive. Cox proportional hazards regression models were utilized for univariate and multivariate analyses to identify prognostic factors. Values of P<0.05 were considered statistically significant. All statistical analyses were performed with R version 4.0.2 (R Foundation for Statistical Computing, Vienna, Austria).