This study was approved by the institutional review board of Harbin Medical University, and consent was obtained from all of the patients in written form. Patients with pathologically confirmed rectal adenocarcinoma who were treatment-naïve were screened in this research, and were examined through preoperative rectal MRI at the Second Afflict Hospital of Harbin Medical University from July 2011 to July 2014. Patients who were found to have a synchronous regional lymph node and/or distant metastases at the initial staging were excluded from the study. All chosen samples were adopted from the crucial cores of histopathologically confirmed carcinomas during the initial surgeries. Two pathologists performed diagnosis on all of the lesions and the findings were reviewed by an expert colorectal cancer pathologist independently; additionally, the stage of tumor was determined according to the system of the International Union Against Cancer.

The MRI examination of patients with RC was carried out using a 3.0 T Philips Achieva TX (Philips Medical Systems, Holland) system with an 8-channel body phased array coil. Each rectal magnetic resonance examination was performed as follows: To decrease the colonic motility, an intramuscular injection of 20 mg of scopolamine butylbromide (Buscopan; Boehringer Ingelheim, Germany) was administered prior to the MRI. Approximately 100-120 mL of saline was rectally administered using an enema syringe. High-resolution magnetic resonance images were obtained, including high-resolution oblique axial T2-weighted turbo spin-echo, oblique coronal T2-weighted turbo spin-echo and sagittal T2-weighted turbo spin-echo coupled with the following parameters: TE (echo time) 81 ms-185 ms, TR (repetition time) 3900-5600 ms, thickness of 3 mm, spacing of 3 mm, matrix of 256 × 256 to 320 × 320, field of view of 250 × 250 to 199 × 199, and echo train length of 17-35.

All magnetic resonance images required at least three abdominal radiologists to analyze the images separately with no clinical information about the patients. The MRI analysis involved tumor morphology, the presence of EMVI, circumferential resection margin, lymph node involvement and tumor stage. The EMVI grading score was adopted from the system proposed by Smith et al[10]. According to this scoring system, scores of 0 to 2 were defined as EMVI-negative disease, and scores of 3 and 4 corresponded to EMVI-positive disease. A score of 3 EMVI included a tumor with a vein that did not change its contour and may have only slightly expanded the vessel. A score of 4 EMVI described an irregularly expanded vein, which indicated the vessel wall was invaded. To achieve a consensus agreement, EMVI negative cases were re-evaluated together by three abdominal radiologists.

To select the study samples, we employed a usual histological categorization according to the WHO classification of tumors. A 3-tiered histological grading system played an important role in this research. The tumor-node-metastasis stage was evaluated, based on the guideline of the 2002 International Union Against Cancer classification. Monoclonal mouse anti-human c-MYC, PCNA and TIMP-1 antibodies were introduced from Santa Cruz Biotechnology (United States). The slides were deparaffinized, rehydrated, immersed in 3% hydrogen peroxide solution for 10 min, heated in citrate buffer (pH 6.0) for 25 min at 95 °C, and cooled for 60 min at room temperature. The slides were washed with PBS (pH 7.4) between each incubation step. Then, the slides were incubated separately with the primary antibodies. Immunoperoxidase staining was performed using the 2-step Envision Method (DAKO, Denmark) abiding by the manufacturer’s instructions, and visualized with 3,3 9-diaminobenzidine tetrachloride (Sigma, United States). Negative controls were slides with no primary antibodies.

Cytoplasm and membrane staining were mensurated for the TIMP1 antibody, and c-MYC and PCNA staining showed nuclear localization. Two pathologists counted the positive cells. The scores of the results according to the immunohistochemical staining were obtained with the examiners being completely ignorant of any information related to the patient’s clinical data. When it comes to clinicopathological association, the researchers applied a 4-tiered scoring system (negative to 3+), taking into consideration the proportion of positive cells and staining intensity as represented before[11].

The statistical analyses were carried out via the SPSS software program (standard version 19.0; SPSS, United States). The distinctions between groups were determined according to the Mann-Whitney U test or the Kruskal-Wallis H test. The correlation among categorical data underwent statistical analysis using the χ² test. Kaplan-Meier curves resulted from the log-rank test for survival analyses. The clinical end point in the research, the period from cancer confirmed to death starting at RC or last contact, was overall survival. Multivariate Cox proportional hazards regression analysis served in the detection of independent prognosticators, which had a conspicuous influence on patient survival. A difference was regarded to be significant under the condition that the P-value was less than 0.05.

A total of 329 RC specimens were collected from patients who underwent an operation. The median patient age was 62 years (range, 21-82 years), and the subjects included 122 (37.1%) females and 207 (62.9%) males. All patients underwent curative resection with tumor-free margins. Among all patients, 99 underwent adjuvant chemotherapy. All tumors were histologically diagnosed, graded, and grouped as lowly and moderately differentiated (n = 319) or highly differentiated (n = 10). The follow-up interval ranged from 1 to 58 mo from the date of diagnosis.

There were 53 cases (16.1%) of EMVI-positive RCs on the initial preoperative MRI scans. Of them, 30 patients (56.6%) had an EMVI score of 3, and 23 patients (43.4%) had an EMVI score of 4. Score 3 MRI-detected EMVI only occurred in small vessels. Score 4 was only observed in medium and large vessels, which was consistent with previous findings[12].

For TIMP1, positive staining was found mainly in the cytoplasm and membrane of the neoplasm cells, and nuclear localization of c-MYC and PCNA was generally observed. Immunostaining of the three proteins was low in some samples but strong in others. Representative examples of IHC expression of TIMP1, c-MYC, and PCNA performed in the study cohorts are shown in Figure 1.

Open in New Tab   Full Size Figure   Download Figure

Figure 1 Representative examples of extramural vascular invasion and immunohistochemistry images in distinct prognostic groups. Overexpression of c-MYC and TIMP1 is a marker of better prognosis, whereas PCNA is a marker of poor prognosis, which was consistent with known biological roles.

There were no significant differences concerning age, sex, chemotherapy and tumor location. However, a significant association was detected between the expression levels of PCNA, TIMP1 and pT (P < 0.01; P = 0.045). Moreover, the overexpression of TIMP1 was associated with the histological grading and perineural invasion (P < 0.01; P < 0.01). Table 1 shows the relationships between the clinicopathological features and protein expression levels.

Due to the fact that it illustrated that the three-protein panel and MRI-detected EMVI was of significant predictive value for survival in the long run, multivariate Cox regression analysis was implemented to decide whether these parameters supplied further information of prognosis independent of known clinicopathologic features which could affect the prognosis. As summarized in Table 2, each parameter had a prognostic significance independently (P < 0.05). Additionally, the three-protein panel displayed greater significance as an independent prognostic factor (HR = 2.110, 95%CI: 1.631-2.556, P < 0.01). The panel combined with the EMVI status could rank patients in a more accurate way (P < 0.01) (Figure 2).

Open in New Tab   Full Size Figure   Download Figure

Figure 2 Immunohistochemistry panel combined with magnetic resonance imaging-detected extramural vascular invasion enhanced the accuracy of prognoses for patients with rectal carcinoma. Combination of EMVI and the IHC panel separated patients into distinct prognostic groups (P < 0.01). EMVI: Extramural vascular invasion; IHC: Immunohistochemistry.

Non-advanced rectal cancer (RC) can be treated using various strategies. Accuracy of preoperative staging is vital for modern management of RC. To accurately classify prognostic risks, this study set out to identify a small, clinically applicable immunohistochemistry (IHC) panel that could be combined with magnetic resonance imaging (MRI)-detected extramural vascular invasion (EMVI) for preoperative prognostic assessment of RC patients.

MRI is important for the local staging of rectal cancer, the patients who showed MRI-detected EMVI positivity had worse survival outcomes than the EMVI-negative cases. Few prior reports contain analyses of the combination of imaging evidence of EMVI and biomolecular factors in RC. The results of this study suggest that the three-protein panel of c-MYC, PCNA and TIMP1 combined with MRI-detected EMVI could provide additional prognostic information for preoperative treatment of RC.

In this study, c-MYC, PCNA and TIMP1 were detected with high expression in 35.9%, 23.7% and 58.7% of tumors, respectively. Significant associations were found between the expression of these proteins and the prognosis (P = 0.032, 0.003, 0.021). Applying these three proteins as an IHC panel could be used to classify patients into different subgroups (P < 0.01). Upon adjustment for known survival covariates, including EMVI, the IHC panel remained an independent prognostic factor. The combination of EMVI and the IHC panel could separate patients into distinct prognostic groups (P < 0.01).

These findings suggest that the three-protein panel of c-MYC, PCNA and TIMP1 combined with MRI-detected EMVI could provide additional prognostic information for preoperative treatment of RC.

EMVI is defined as the presence of malignant cells within the blood vessels beyond the muscularis propria of rectal cancer.

This is an excellent study about the combination of three-gene immunohistochemical panel and MRI-detected EMVI to assess prognosis in non-advanced rectal cancer patients.