Bioinformatics analysis: The UALCAN platform was used to access MICA mRNA levels in normal (uninvolved) colon and CRC tissues [19]. This resource for gene expression analysis uses data from The Cancer Genome Atlas (TCGA). mRNA data are expressed as transcripts per million and representative of standard deviations from the median across samples for the given cancer type. PROGgeneV2, prognostic database [20], was used to perform Kaplan Meier and proportional hazards survival analyses in CRC patients associated to mRNA levels of MICA (GSE41258 and GSE29621 independent publicly available data sets).
Patients and tissue samples: The study population was derived from the University of Mississippi Medical Center (UMMC), Jackson, MS, USA. Specimens collected (2006-2016) following surgery were de-identified and later provided a unique study identification. Clinical and pathological characteristics of study subjects are provided in Table 1.
Table 1: Clinicopathological characteristics of patients
|
Characteristic
|
Finding
|
Age, years, mean (range)
|
59.2 (23-87)
|
Sex, Number (%)
|
|
Male
|
50 (52.1%)
|
Female
|
46 (47.9%)
|
Race/ethnicity, number (%)
|
|
African Americans
|
56 (58.3%)
|
Non-Hispanic Whites
|
40 (41.7%)
|
Site, Number
|
|
Colon
|
62 (64.6%)
|
Rectum
|
34 (35.4%)
|
TNM stage, number (%)
|
|
I
|
11 (11.4%)
|
II
|
30 (31.3%)
|
III
|
35 (36.5%)
|
IV
|
20 (20.8%)
|
Histological grade, number (%)
|
|
Well-differentiated
|
6 (6.3%)
|
Moderately differentiated
|
78 (81.3%)
|
Poorly differentiated
|
7 (7.3%)
|
Unknown
|
5 (5.1%)
|
Lymph node metastasis, number (%)
|
|
Negative
|
36 (37.5%)
|
Positive
|
50 (52.1%)
|
Unknown
|
10 (10.4%)
|
Surgical margins, number (%)
|
|
Negative
|
74 (77.1%)
|
Positive
|
18 (18.8%)
|
Unknown
|
4 (4.1%)
|
Follow-up time (years), median (range)
|
4.6 (0.1-10.3)
|
The data includes sex, race, TNM stage, histological grade, evidence of LNM, surgical margins, survival times, and status. Tumor and normal colonic tissues, adjacent to tumor, were obtained immediately after operation. We included 96 cases, assessed by a board-certified pathologist (VS). Staging was performed according to the guidelines of the American Joint Committee on Cancer. Following surgery, clinical follow-up data were obtained, with the median follow-up of 5.4 years (range 0.1-10.3 years) for the 96 patients. Due to its retrospective nature, the Institutional Review Board waived the need for informed consent. The study was approved by the University of Mississippi Medical Center (UMMC) Institutional Review Board under protocol # 2012-0205. All methods were performed according to standards set by the Declaration of Helsinki.
This study (under Institutional Review Protocol number 2012-0205) was performed according to standards set by the Declaration of Helsinki.
Construction of tissue microarrays: Tumor stage-matched tissues were used to create tissue microarrays (TMA). For each patient, representative formalin-fixed paraffin-embedded (FFPE) tissue blocks including a normal block and a tumor block. A total of 192 samples for the TMA construction were included in the final composite block. Based on the verified histological features, FFPE blocks of primary tumors were selected by the pathologist. From the primary FFPE blocks, cylindrical cores of 2-mm diameter were transferred to paraffin blocks using a Beecher MTA1 Manual Tissue Arrayer (Beecher Instruments, Sun Prairie, WI). For IHC staining and analysis, the resulting TMA composite blocks were sectioned at 5-μm thickness.
Immunohistochemistry: As described before [9, 21], IHC was performed according to manufacturer’s instructions provided in ABC Kit (Vector Laboratories Inc., Burlingame, CA). Following antigen retrieval, with a citrate buffer (pH 6.0) for 20 min, and incubation with 3% hydrogen peroxide, the FFPE TMA sections were deparaffinized and rehydrated during 10 min. To block unspecific binding, the slides were treated with Protein Block Serum-Free (Cat X0909, Dako, Santa Clara, CA) for 12 min followed by incubation with 10% normal serum for 1 h at room temperature. Next, the TMA slides were incubated with rabbit anti-human primary polyclonal antibody against MICA in 1:25 dilution (Cat# PA5-35346, Thermo Scientific, Waltham, MA) overnight at 4°C. Next, the slides were washed with phosphate-buffered saline (PBS), incubated with components of the ABC kit, and with 3, 3-diaminobenzidine (DAB) for color development. Slides were counterstained in hematoxylin and mounted. Subcellular localizations of MICA were defined as cytoplasmic/membranous or globular staining by the pathologists, and scored. Evaluation of IHC was independently performed by two independent evaluators blinded to the specific diagnosis or prognosis for each individual case. To assess the MICA cytoplasmic staining intensity, a modified version of the “quickscore” method was utilized [9]. Data are expressed as median (interquartile range). To assess the association between MICA expression and clinical features in the CRC cases, patients were dichotomized by low and high MICA tumor expression, based on the optimal cutoff point calculated as the value with the most significant log‑rank test split (3.4 for combined intensity score).
Statistical analysis: The SPSS software package, version 13.0 (SPSS Inc., Chicago, IL USA), SAS 9.4 (SAS Inc., Cary, NC, USA) and GraphPad Prism (GraphPad Software, La Jolla, CA were used to analyze the data. The difference in MICA gene expression between uninvolved tissue and tumor tissue or for any other pairwise comparison obtained using bioinformatics analyzes was evaluated by Student's t‑test. One‑way ANOVA and Dunnett's multiple comparisons were utilized when three or more groups were compared. Pairwise comparisons were always relative to normal tissue. For IHC data, differences were compared by Mann‑Whitney U test for non‑matched data or Wilcoxon signed rank test for matched‑pairs. Two‑sided P‑values were determined via Chi‑square or Fisher's exact tests for categorical variables. Overall survival was analyzed by the Kaplan‑Meier and proportional hazards methods with the use of the log‑rank test and hazard risks (HR) and 95% confidence intervals (95% CI) to compare overall survival. For all analyses, the level of significance was set at P<0.05.