The patient medical charts and pathology reports were retrieved for identifying surgically resected and pathologically confirmed ESCC cases. In total, 78 ESCC cases were obtained between 2000 and 2018. Based on the hematoxylin and eosin (H&E) slide review and chart review, only patients with primary esophageal cancers who were chemotherapy- or radiation therapy-naive at the time of the surgery, and whose formalin-fixed, paraffin-embedded (FFPE) tumor tissue blocks and complete medical charts were available were included in this study. Fourteen patients were excluded from the study. In total, 64 patients with ESCC who underwent radical esophagectomy (Ivor-Lewis operation) with standard lymphadenectomy as the initial definitive treatment were included in the study.
The clinical characteristics and follow-up data were retrospectively obtained from the medical records, pathology report files, and radiological study results. Based on the clinician’s judgment, the type of esophageal resection was determined and the selected patients were offered adjuvant chemotherapy with or without radiation therapy. The radiation doses ranged from 5000 cGy to 5400 cGy (23 dose fractions) over a period of 8 weeks. The patients underwent fluoropyrimidine-/taxene-based chemotherapy. Chemotherapy was prescribed based on the performance statuses, comorbidities, and toxicity profiles of individual patients.
Smoking history was measured in packs per year and the patients were classified into 2 categories using 20 packs/year as the cut-off value with heavy smoking defined as > 20 packs/year [13]. Alcohol consumption status was divided into 2 categories using 14 drinks/week as the cut-off value and heavy alcohol consumption was defined as > 14 drinks/week [13].
The pathology reports and histological slides were reviewed and were re-evaluated by an experienced gastrointestinal pathologist (MJK). The diagnosis and histological differentiation were evaluated according to the World Health Organization classification [14]. The stage of the esophageal carcinomas was determined based on the American Joint Committee on Cancer staging system (Eighth edition) [13].
The degree of infiltration of TILs was evaluated from the H&E-stained slides and scored from 0 to 3 as follows: 0, none; 1+, focal infiltration with more than mild staining intensity; 2+, moderate infiltration with more than mild staining intensity; 3+, diffuse infiltration with more than mild staining intensity.
The study protocol was approved by Hallym University Sacred Heart Hospital Institutional Review Board (IRB No. 2019-03-014-001) and performed in accordance with the relevant guidelines and regulations (Declaration of Helsinki). Informed consent was obtained from the patients and from the next of kin (deceased patients) before enrollment in the study.
Immunohistochemistry andmicrosatellite status determination
Immunohistochemical staining was performed on 4-μm thick tissue microarray (TMA) sections using the BenchMark XT automated immunostainer system (Ventana Medical Systems, Inc., Tucson, AZ, USA), according to the manufacturer’s instructions. The primary antibody used in this analysis was anti-PD-L1 (rabbit anti-human PD-L1 monoclonal, 1:25, clone SP142; Ventana). PD-L1 expression was evaluated based on the proportion of membranous staining in the tumor cells. The expression was scored as follows: 0 for <5% of tumor cells, 1+ for 5-10%, 2+ for 10-50%, and 3+ for >50% of tumor cells. Consistent with several previous published reports, an IHC score of ≥1+ was considered positive [11,15]. To evaluate the expression of MMR proteins, the following primary antibodies were used in this analysis: anti-MLH1 (pre-diluted; Ventana Medical Systems), anti-MLH2 (1:300; Cell Marque, Rocklin, CA, USA), anti-MSH6 (1:200; Cell Marque), and anti-PMS2 (pre-diluted; Ventana Medical Systems) antibodies.
MSI status in the tumor was analyzed by immunohistochemistry of MMR proteins and by multiplex polymerase chain reaction (PCR) for five quasi-monomorphic mononucleotide repeat markers (BAT25, BAT26, NR21, NR24, and NR27). The MSI/dMMR tumors were defined as those exhibiting loss of expression of one or more MMR proteins or exhibiting high-level MSI (MSI-H), which was determined by PCR [16,17]. Microsatellite-stable/proficient MMR (MSS/pMMR) tumors exhibited intact MMR protein expression and/or MSS or low-level MSI (MSI-L) status.
Mutation and HPV detection analyses
Genomic DNA was extracted from 10-µm thick sections of 10% neutral manually dissected FFPE tumor tissue using the Maxwell® 16 FFPE Purification Kit for DNA (Promega, USA). The mutations in the KRAS (exon 2 and 3), BRAF (exon 14), and PIK3CA (exon 9 and 20) genes were analyzed by directional sequencing of PCR fragments amplified from the genomic DNA as previously described [11]. All sequences were confirmed in duplicate for replicate amplification reactions.
The HPV status was determined using the PANA RealTyperTM HPV Kit (PANAGENE, Daejeon, South Korea), according to the manufacturer’s instructions. This kit, which is approved for clinical use in Korea, detects 40 HPV genotypes, including 20 high-risk genotypes (16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 69, 70, 73, and 82), 2 low-risk genotypes (6 and 11), and 18 other genotypes (30, 32, 34, 40, 42, 43, 44, 54, 55, 61, 62, 67, 74, 81, 83, 84, 87, and 90).
Statistical analysis
The clinical and pathological parameters are represented as mean ± standard deviation for continuous variables and as frequency for categorical variables. The categorical variables were analyzed using the chi-squared (χ2) test or two-sided Fisher’s exact test. Survival analyses were performed using the Kaplan-Meier method. The survival curves were compared using the log-rank test. Overall survival (OS) was defined as the interval from the first day of surgery until death or the end of the follow-up period. Disease-free survival (DFS) was defined as the interval from the first day of surgery until tumor progression, death, or end of the follow-up period. We also used the Cox proportional hazards model for the univariate and multivariate analyses of OS and DFS. OS and DFS were analyzed until February 2019. All statistical analyses were performed in the SPSS statistical analysis software (v18; SPSS, Chicago, IL, USA). The difference was considered statistically significant when the P-value was less than 0.05.