Smad4 Status and 5-FU-Based Treatment in Colorectal Cancer
Smad4 Status and 5-FU-Based Treatment in Colorectal Cancer
Tumour samples from 251 consecutive patients with upfront CRC resection between 2005 and 2009 treated at the Stanford Cancer Institute were included in this retrospective review. Specifically excluded from this study were patients who received neoadjuvant therapy (n=9). One patient was excluded because of in situ disease. A total of 241 patients were included in our final analysis.
Clinicopathological records were obtained for all patients from the Stanford Hospital and Clinics electronic medical record system. The following information was obtained: type of initial surgical procedure and subsequent therapy, tumour size, grade and location, margin status and the presence or absence of lymph node metastases. Clinical follow-up data were obtained by reviewing patient records and using information available online from the Social Security Death Index (http://www.genealogybank.com). Follow-up status on living patients was collected through December 2012. All data were obtained under the approval of the Stanford University Institutional Review Board.
H&E-stained slides from each primary tumour were reviewed by a single pathologist (TL) to confirm the diagnosis of CRC. The following histological features were recorded for each: histological subtype, grade, extent of invasion and presence or absence of lymph node metastases. Each tumour was assigned a stage according to the American Joint Committee on Cancer TNM staging criteria.
Triplicate tissue cores from 251 resected primary colon tumours and matched normal controls were used to construct TMAs. Four TMAs containing a total of 196 tissue cores, each measuring 1.0 mm, were created using a tissue arrayer (Beecher Instruments, Silver Spring, Maryland, USA). Representative areas from each case were selected from the microarray from paraffin blocks based on H&E-stained sections (figure 1).
(Enlarge Image)
Figure 1.
A representative photomicrograph of normal colon (A) versus adenocarcinoma (B) (original magnification ×400). Each patient was represented by two cores of tumour tissue alongside one core of matched normal control.
Expression of Smad4 was assessed using immunohistochemistry (IHC) on 4-μm-thick sections of paraffin-embedded tissue from each TMA. Sections were heated at 60°C for 60 min, deparaffinised in xylene and rehydrated through a graded alcohol series. Three per cent H2O2 block was used to quench endogenous peroxidase activity. Antigen retrieval was performed in a pressure cooker using a Borg Decloaker RTU antigen retrieval solution (Biocare Medical). Sections were then blocked in normal horse serum (Vector) and incubated in a humidified chamber overnight at 4°C with a 1:200 dilution of primary anti-Smad4 antibody (sc-7966, Santa Cruz). Slides were washed in phosphate buffered saline and incubated in antimouse secondary antibody (Vector) for 30 min at room temperature. Staining was visualised using DAB (Vector) at room temperature. Slides were then counterstained with haematoxylin, rinsed in water and dehydrated through a graded alcohol series and xylene, and mounted with VectaMount (Vector).
All slides were reviewed by two pathologists (TL and RKP) blinded to treatment outcomes and scored on a scale of 0–3 for intensity of staining—absence of Smad4 staining (0), indeterminate (1), faint-to-moderate (2) and prominent nuclear staining (figure 2). Scores from triplicate cores were averaged and used for subsequent analysis. Cores scored as 2 and 3 were deemed 'positive', and cores scored as 0 were deemed 'negative'. Cores scored as 1 were excluded. Smad4 protein expression was subsequently analysed by positive versus negative staining.
(Enlarge Image)
Figure 2.
Smad4 immunohistochemistry staining of colorectal tumours demonstrates (A) no staining, (B) faint-to-moderate staining and (C) prominent nuclear staining (original magnification ×400).
Overall survival (OS) was defined as the time of surgery to the date of death from any cause. Disease-free survival (DFS) was defined as the date of surgery to the date of tumour recurrence at any site or to the date of last follow-up.
OS and DFS were analysed using Kaplan–Meier and log-rank tests. Univariate analyses were performed to evaluate age (≤ vs >66 years), gender (male vs female), tumour stage (1–4), presence of lymph node metastases (positive vs negative), overall stage (I–IV), tumour grade, location and margin status (positive vs negative) and Smad4 expression (positive vs negative). Fisher's exact test was used to compare patients with Smad4-positive versus Smad4-negative expression. Significance was determined as a p value <0.05.
The data were analysed in a multivariate analysis with Cox proportional hazards regression using clinical characteristics with a p value <0.05 in the univariate analysis to identify characteristics prognostic for worse outcomes. HRs were reported with a 95% CI. All statistical analyses were performed using Statistical Analysis Software (SAS Institute, Cary, North Carolina, USA). A two-sided p value of <0.05 was considered to be significant.
Materials and Methods
Patient Samples
Tumour samples from 251 consecutive patients with upfront CRC resection between 2005 and 2009 treated at the Stanford Cancer Institute were included in this retrospective review. Specifically excluded from this study were patients who received neoadjuvant therapy (n=9). One patient was excluded because of in situ disease. A total of 241 patients were included in our final analysis.
Clinicopathological records were obtained for all patients from the Stanford Hospital and Clinics electronic medical record system. The following information was obtained: type of initial surgical procedure and subsequent therapy, tumour size, grade and location, margin status and the presence or absence of lymph node metastases. Clinical follow-up data were obtained by reviewing patient records and using information available online from the Social Security Death Index (http://www.genealogybank.com). Follow-up status on living patients was collected through December 2012. All data were obtained under the approval of the Stanford University Institutional Review Board.
H&E-stained slides from each primary tumour were reviewed by a single pathologist (TL) to confirm the diagnosis of CRC. The following histological features were recorded for each: histological subtype, grade, extent of invasion and presence or absence of lymph node metastases. Each tumour was assigned a stage according to the American Joint Committee on Cancer TNM staging criteria.
Tissue Microarray
Triplicate tissue cores from 251 resected primary colon tumours and matched normal controls were used to construct TMAs. Four TMAs containing a total of 196 tissue cores, each measuring 1.0 mm, were created using a tissue arrayer (Beecher Instruments, Silver Spring, Maryland, USA). Representative areas from each case were selected from the microarray from paraffin blocks based on H&E-stained sections (figure 1).
(Enlarge Image)
Figure 1.
A representative photomicrograph of normal colon (A) versus adenocarcinoma (B) (original magnification ×400). Each patient was represented by two cores of tumour tissue alongside one core of matched normal control.
Immunohistochemistry
Expression of Smad4 was assessed using immunohistochemistry (IHC) on 4-μm-thick sections of paraffin-embedded tissue from each TMA. Sections were heated at 60°C for 60 min, deparaffinised in xylene and rehydrated through a graded alcohol series. Three per cent H2O2 block was used to quench endogenous peroxidase activity. Antigen retrieval was performed in a pressure cooker using a Borg Decloaker RTU antigen retrieval solution (Biocare Medical). Sections were then blocked in normal horse serum (Vector) and incubated in a humidified chamber overnight at 4°C with a 1:200 dilution of primary anti-Smad4 antibody (sc-7966, Santa Cruz). Slides were washed in phosphate buffered saline and incubated in antimouse secondary antibody (Vector) for 30 min at room temperature. Staining was visualised using DAB (Vector) at room temperature. Slides were then counterstained with haematoxylin, rinsed in water and dehydrated through a graded alcohol series and xylene, and mounted with VectaMount (Vector).
All slides were reviewed by two pathologists (TL and RKP) blinded to treatment outcomes and scored on a scale of 0–3 for intensity of staining—absence of Smad4 staining (0), indeterminate (1), faint-to-moderate (2) and prominent nuclear staining (figure 2). Scores from triplicate cores were averaged and used for subsequent analysis. Cores scored as 2 and 3 were deemed 'positive', and cores scored as 0 were deemed 'negative'. Cores scored as 1 were excluded. Smad4 protein expression was subsequently analysed by positive versus negative staining.
(Enlarge Image)
Figure 2.
Smad4 immunohistochemistry staining of colorectal tumours demonstrates (A) no staining, (B) faint-to-moderate staining and (C) prominent nuclear staining (original magnification ×400).
Statistical Analysis
Overall survival (OS) was defined as the time of surgery to the date of death from any cause. Disease-free survival (DFS) was defined as the date of surgery to the date of tumour recurrence at any site or to the date of last follow-up.
OS and DFS were analysed using Kaplan–Meier and log-rank tests. Univariate analyses were performed to evaluate age (≤ vs >66 years), gender (male vs female), tumour stage (1–4), presence of lymph node metastases (positive vs negative), overall stage (I–IV), tumour grade, location and margin status (positive vs negative) and Smad4 expression (positive vs negative). Fisher's exact test was used to compare patients with Smad4-positive versus Smad4-negative expression. Significance was determined as a p value <0.05.
The data were analysed in a multivariate analysis with Cox proportional hazards regression using clinical characteristics with a p value <0.05 in the univariate analysis to identify characteristics prognostic for worse outcomes. HRs were reported with a 95% CI. All statistical analyses were performed using Statistical Analysis Software (SAS Institute, Cary, North Carolina, USA). A two-sided p value of <0.05 was considered to be significant.
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