br The current study aimed to
The current study aimed to more broadly investigate the role of CDX2 status as well as other important markers of co-lonic differentiation of potential prognostic value. In tissue sections, Clozapine N-oxide of CDX2 is patchy (Fig. 1) and potentially can lead to misleading results from a tissue microarray–based experiment. Therefore, to develop a means to address the known variability in expression, we designed a retrospective study looking at CDX2 expression in stage II colon cancers using multiple blocks of whole-slide immunohistochemistry. We also explored the expression of Muc2, CDX1 GPX2, and villin to assess whether these might better predict outcome in CRC.
2. Materials and methods
2.1. Patient selection and case review
Institutional research ethics board approval for retrospec-tive review and immunohistochemical testing was obtained. Pathology archives were searched for cases of colon cancer be-tween 2006 and 2013. Rectal cancers were excluded because the effects of neoadjuvant therapy on CDX2 immunohisto-chemistry were not clear. Cases with less than 3 years of fol-low-up were excluded. Pathology reports were reviewed to select only stage II cases. The electronic charts were then reviewed, and cases that received adjuvant chemotherapy were excluded. In total, 210 cases of colon cancer were included in the study. The pathology reports and all tumor slides were reviewed by M. J. C. and one of the other pathologists (D. K. D., J. C. W., J. P., or S. C.). Three blocks of tumor were se-lected for CDX2 staining in each case that included represen-tative histology, the deepest point of invasion, and any high-grade component or area of tumor with a distinct histology.
Figure 1 Low-power image of CDX2 immunohistochemistry with inset images of high-magnification (original magnification ×200) images showing distinct staining patterns observed in separate foci of the tumor.
Elastic stains were used routinely in most cases to aid in iden-tifying venous invasion. Patient outcome was determined by manual review of the chart (by M. J. C. and R. C.) with survival calculated from the date of surgery to the date of cancer-related death or last follow-up appointment. To screen other markers of colonic differentiation, we selected a subset of 11 patients who died of CRC compared with the next historical case of a CRC patient who did not succumb to disease during the study period as a control. This subset was expanded for Muc2 to study the role of Muc2 expression in a larger cohort of cases.
Immunohistochemistry was performed on slides cut from paraffin-embedded blocks at 4 μm and dried at 60°C for 45 minutes and 45°C overnight. Staining was performed using the EnVision Flex System on the Autostainer Link 48 platform (Dako Santa Clara, CA). Antigen retrieval was completed in a 97°C water bath at pH 5.9 to 6.3 for 20 minutes, peroxidase block for 5 minutes, primary antibody for 20 minutes, mouse/rabbit linker for 15 minutes, horseradish peroxidase for 20 minutes, and diaminobenzidine for 10 minutes. Anti-bodies for CDX2 DAK-CDX2 (Dako [IR080]; ready to use) CDX2–88 (AbCam [ab157524]; 1:50 dilution), CDX1
2.3. Gene expression from The Cancer Genome Atlas database
The provisional database of colon cancer cases was accessed on August 5, 2018, through cbioportal.org, and 382 cases with RNA Seq data available were queried for cases with reduced Muc2 and CDX2 expression [33,34]. To detect cases with lower levels of Muc2 and CDX2, a cutoff of cases less than 0.4 SD from the mean was used.
As demonstrated by the patchy staining of CDX2 in Fig. 1, it can be difficult to assess the CDX2 status of colon cancers from whole-slide immunohistochemistry. There are often
CDX2 and Muc2 in stage II colon cancer 73
Figure 2 Semiquantitative immunohistochemistry scoring system (original magnification ×200).
focal areas in tumors that show loss of expression for CDX2 despite most of the tumor being positive. It is not clear from current studies how to score these cases. To address the vari-ability, our approach used staining of multiple blocks for CDX2 in an effort to better understand the variability and iden-tify a robust means to implement CDX2 status into clinical practice.
In total, we studied 210 cases from patients with an average age at diagnosis of 74 years, with an approximately equal split between male and female patients (Table). Most cases were right sided (69%). All cases were stage II lymph node nega-tive. Most cases were T3, with 12% of cases staged as T4a or T4b. On average, 22 lymph nodes were identified. Most cases were low grade (94%). Lymphovascular invasion (small vessel invasion) was identified in 18% of cases, and venous in-vasion was reported in 25% of cases. In other series, venous in-vasion has been typically reported in the range of 30% to 40% with the routine use of elastic stains [35-37]. Our observed lower rate may reflect that our study was limited to patients with early stage disease and a small subset of our older cases predated the routine use of elastic stains for identification of venous invasion.