From April 2008 to March 2019, 320 consecutive patients who underwent surgical resection for UC were collected from Yokohama City University Medical Center in Japan. The exclusion criterion was no data on serum anti-p53 Abs before the operation. A total of 250 patients were analyzed in this study. We divided the patients into two groups: Group non-CAC included those who underwent surgery for severe or intractable colitis and had not been diagnosed with carcinoma or dysplasia histologically by a biopsy or surgical specimen, and Group CAC included those who had been diagnosed with carcinoma or dysplasia. Of these 250 UC patients, 219 (87.6%) were in Group non-CAC, and 31 (12.4%) were in group CAC (Fig. 1). The history of other cancer, smoking habit and family history of colorectal cancer (CRC) were reviewed. The clinicopathological features of UC, such as the age at the operation, duration of disease, extension of disease and medications, were collected from the medical records.
An enzyme-linked immunosorbent assay (ELISA) for Anti-p53 Abs and conventional tumor makers
An ELISA for the detection of anti-p53 Abs in serum was carried out with a commercially available ELISA kit (MESACUP anti-p53 test; Medical & Biological Laboratories, Nagoya, Japan). In brief, serum samples and calibrators were added to each well of a microwell plate coated with wild-type human p53 or control protein. Samples were incubated on the plate for 60 min (20-30 ℃). After washing, a peroxidase-conjugated anti-human IgG anti-p53 Abs was added, followed by further incubation for another 60 min (20-30 ℃). After washing again, the substrate was added, and the plate was incubated again for 30 min (20-30 ℃). A stop solution was then added to each well to stabilize the color development. The value in each sample was determined by comparing the optical density of the sample to that of the anti-p53 standard. The cut-off value was 1.3 U/ml, according to a previous colorectal cancer study12, 14, 15.
The carcinoembryonic antigen (CEA) levels were measured with a CEA-2 enzyme immune assay (EIA) kit (Elecsys CEAII; Roche Diagnostics K.K., Tokyo, Japan) following the manufacturer’s instructions with a cut-off value of 5.0 ng/ml. The cancer antigen 19-9 (CA19-9) levels were measured with a CA19-9 EIA kit (Elecsys CA19-9; Roche Diagnostics K.K.) with a cut-off value of 37 U/ml.
For those patients revealed to have dysplasia or CRC by colonoscopy, we also evaluated the p53 status with immunohistochemistry using biopsied or surgical specimens. In brief, parafﬁn-embedded tissue samples were cut into serial sections of 3-4 µm thickness, placed on coated slides and deparafﬁnized through a series of xylene and ethanol baths. The slides were then incubated with anti-p53 primary antibodies (DO-7; DAKO, Glostrup, Denmark), followed by biotin-conjugated goat antibody against mouse as a secondary antibody (E0433; DAKO). The following steps were performed using a standard ABC method (Elite ABC kit, Vectastain; Vector Laboratories, Burlingame, CA, USA): 3,3-Diaminobenzidine was used as substrate for the peroxidase enzyme reaction (Vectastain; Vector Laboratories), and all slides were counterstained with hematoxylin and observed under a microscope (CH40; Olympus, Tokyo, Japan).
In this study, we defined immuno-suppressive therapy as a patient with a medical history of receiving prednisolone exceeding 20 mg per day for the equivalent of 2 weeks or more, immunosuppressant drugs (including cyclosporine and tacrolimus), immunomodulator drugs (including 6-mercaptopurine, azathioprine and methotrexate) or biologic therapies (including infliximab and adalimumab) for UC treatment. According to a previous report, these therapies induced an immunocompromised state in inflammatory bowel disease patients.
Differences between categorical variables were tested using Pearson’s chi-squared test, and differences between continuous variables were tested using the Mann-Whitney U test. Statistical analyses were performed using the SPSS statistical software program, version 22.0 (SPSS Inc., Chicago, IL, USA). All tests were 2-sided, and values less than 0.05 were considered statistically significant.