Patients
Two TMAs with a total of 1800 CRC samples were included in this study. The first TMA was manufactured from surgical specimens of 1420 CRC patients at the Institute of Pathology of the University Hospital of Basel, while the second included 380 samples from our institution. Table 1 shows the flow diagram for the study cohort.
All specimens were collected from patients who underwent primary surgery for colorectal cancer without neoadjuvant therapy, so as to evaluate the natural course of the disease. Postoperative therapy was executed according to the guidelines for the treatment of colorectal carcinoma [18]. For both cohorts, survival data and pathological parameters were complete. No data were available regarding disease progression as assessed by clinical or RECIST guidelines.
TMA construction was as described [19]. In brief, hematoxylin and eosin-stained sections were made from each block to define representative tumour regions. Tissue cylinders with a diameter of 0.6 mm were then punched from tumour areas of each “donor” tissue block using a home-made semi-automated precision instrument and brought into empty recipient paraffin blocks. Four µm sections of the resulting TMA blocks were transferred to an adhesive coated slide system (Instrumedics Inc., Hackensack, New Jersey). Patient information and clinical data, such as age, sex, localization and type of tumour, pTNM-stage and carcinoma grade were retrospectively retrieved from clinical and pathological databases (Table 2).
Table 2
Clinicopathological features of CRCs.
Clinicopathological features
|
|
Available, n
|
Gender
|
|
Male
|
690
|
Female
|
675
|
Age
|
Mean: 69 (29–96)
|
Tumour stage
|
|
pT1
|
62
|
pT2
|
219
|
pT3
|
869
|
pT4
|
200
|
Nodal status
|
|
pN0
|
725
|
pN1
|
330
|
pN2/3
|
280
|
Grading
|
|
G1
|
20
|
G2
|
1141
|
G3
|
185
|
Tumour localization
|
|
Right-sided colon
|
270
|
Transverse colon
|
86
|
Left-sided colon
|
315
|
Rectum
|
335
|
Histological type
|
|
Non-mucinous
carcinoma
|
918
|
Mucinous carcinoma
|
84
|
others
|
14
|
In detail, 690 of the patients were male and 675 were female. The distribution of cancers according to tumour size was as follows: 62 pT1 cancers, 219 pT2 cancers, 869 pT3 and 200 pT4 cancers. 725 of the tumours were staged as nodal negative, while 610 specimens revealed nodal involvement. Regarding the tumour grading, 20 tumour samples were classified as G1, 1141 G2 and 185 G3. All tumours were assessed by two experienced colorectal examiners (NM, KG). Follow-up data were obtained from local cancer register boards or via attending physicians. The median follow-up time was 46 months (range 1–152 months) for the first and 36 months (range 1–179 months) for the second cohort.
For statistical analyses, tumour localization was grouped as follows: right-sided cancer (cecum, ascending colon), cancer of the transverse colon including both flexures, cancer of the left-sided colon (descending colon, sigmoid colon) and rectum. The utilization of tissues and clinical data was according to the Hamburger Krankenhaus Gesetz (§ 12 HmbKHG) and approved by our local Ethical Committee.
Immunohistochemistry
Freshly cut TMA sections were analyzed during one day in one single analysis. Slides were deparaffinized and exposed to heat-induced antigen retrieval for 5 minutes in an autoclave at 121°C in pH 7.8 Tris-EDTA-Citrate buffer prior to incubation with antibody PHD1 (polyclonal; rabbit; Novus Biologicals; 1/450 dilution). Bound antibody was visualized using the EnVision Kit (Dako). PHD1 immunostaining was analyzed by one person experienced in IHC analysis (KG). PHD1 staining was predominantly localized in the cytoplasm of the cells and was rarely accompanied by lower expression levels in the nucleus of the cells. PHD1 staining was homogenous in the analyzed tissue samples and staining intensity was semiquantitatively recorded as low or high intensity staining.
Statistics
Statistical calculations were performed with JPM 10 software (SAS Institute Inc., NC, USA). Contingency tables and the chi²-test were performed to search for associations between PHD1 expression and tumor phenotype. Survival curves were calculated according to Kaplan-Meier. The Log-Rank test was applied to detect significant differences between groups, and COX proportional hazards regression analysis was performed to test the statistical independence and significance between pathological, molecular and clinical variables.