Patient population
From January 2005 through December 2016, 95 consecutive patients with CRLM were admitted to the Niigata University Medical and Dental Hospital for surgical intervention. Of these patients, 88 underwent potentially curative hepatectomy and were included in this retrospective study. Participants comprised 59 men and 29 women with a median age of 65 (range, 33–83) years at the time of initial hepatectomy. This study was approved by the ethics committee of Niigata University Graduate School of Medical and Dental Sciences (approval number: 2017-0052). The status of the primary tumor was assessed using the TNM staging system [14]. Synchronous liver metastasis was defined as metastasis detected simultaneously with the primary tumor or found within 1 month after surgery for the primary colorectal tumor; metachronous liver metastasis was defined as metastasis detected later than 1 month after primary colorectal surgery.
Preoperative chemotherapy for CRLM
Of the 88 patients, 30 were administered preoperative chemotherapy for CRLM with a combination of 5-fluorouracil (5-FU) and oxaliplatin or irinotecan. Of the 30 patients who were administered preoperative chemotherapy, 13 were treated with bevacizumab, 3 with panitumumab, and 1 with cetuximab. Tumor response to preoperative chemotherapy for CRLM was evaluated using contrast-enhanced computed tomography and assessed based on the Response Evaluation Criteria In Solid Tumors (RECIST) version 1.1 [15].
Hepatectomy procedures
All metastatic lesions were indicated for surgery when considered resectable and the patients had an acceptable operative risk. In this study, major hepatectomy was defined as the removal of ≥ 3 Couinaud segments and minor hepatectomy was defined as the removal of < 3 Couinaud segments. The hepatectomy procedure was determined according to the hepatic tumor status (number, size, and location), hepatic functional reserve, and patient’s general condition.
Patient follow-up after hepatectomy
As adjuvant chemotherapy after hepatectomy for CRLM, 38 patients received intravenous or oral administration of 5-FU or its derivatives within approximately 1 year. Patients were regularly followed-up with physical and blood biochemistry examinations and imaging investigations every 3–6 months after hepatectomy for CRLM. The median follow-up after hepatectomy was 65.4 (range, 0.2–156.9) months.
Pathologic evaluation
Resected specimens were submitted to the Department of Surgical Pathology at our hospital. Histologic grading of the hepatic tumor was done according to the areas with the highest grade. Hepatectomy margin status was assessed histologically as either R0 (no residual tumor) or R1 (microscopic residual tumor), depending on the absence or presence of histologically verified tumor cells in the hepatectomy margin. Pathologic response to preoperative chemotherapy for CRLM was evaluated based on the Japanese classification of colorectal carcinoma as follows: grade 0, no recognizable cytologic or histologic therapeutic effect was observed; grade 1, viable cells accounted for at least one-third of the tumor tissue; grade 2, viable cells accounted for less than one-third of the tumor tissue; and grade 3, no viable cells were observed.
Immunohistochemistry
Immunohistochemical staining was performed for the surgically resected specimen. A rabbit monoclonal antibody against NQO1 (Epitomics, Burlingame, CA) was used at a dilution of 1:200. Three serial 3-µm sections were recut and prepared from each block: 1 each for hematoxylin-eosin staining, immunohistochemical staining, and negative control. Two independent surgical pathologists blinded to the clinical details assessed each section. Before staining, sections were microwaved for 21 min in 10 mM sodium citrate buffer (pH 6.0). After overnight incubation at 4 °C with NQO1 antibody, sections were incubated with goat anti-rabbit IgG polymerized horseradish peroxidase-labeled secondary antibody (Epitomics) at room temperature for 30 min. Diaminobenzidine was used as the chromogen and sections were counterstained with hematoxylin.
Patterns of NQO1 expression and definition of NQO1 polymorphism status
NQO1 expression was defined as the presence of cytosolic and/or nuclear staining as described previously [9, 16]. Then, according to NQO1 expression in tumor specimens of CRLM, patients were classified as either those with positive expression in CRLM (Fig. 1A) or those with loss of expression in CRLM (Fig. 1B). Additionally, non-neoplastic interlobular biliary epithelial cells of the liver, which typically show immunopositive staining for NQO1 (Fig. 1A and B) [3], occasionally showed no NQO1 immunoreactivity (Fig. 1C), probably because homozygosity for the NQO1 polymorphism is associated with loss of NQO1 protein [11]. Thus, in the current study, patients were classified as follows: patients with NQO1 polymorphism, characterized by no NQO1 expression in non-neoplastic intralobular biliary epithelial cells of the liver, or patients without NQO1 polymorphism, characterized by NQO1-positive expression in non-neoplastic intralobular biliary epithelial cells of the liver.
Statistical analysis
Categorical variables were compared using Fisher’s exact test. The follow-up period was defined as the interval between the date of hepatectomy and last follow-up. Cumulative survival was estimated using the Kaplan-Meier method, and the log-rank test was applied to compare survival between the groups. To identify independent prognostic factors, the Cox proportional hazards regression model was used. All statistical evaluations were performed using the PASW Statistics 23 software package (SPSS, Inc., Chicago, IL). All tests were two tailed and p < 0.05 was considered statistically significant.