A series of 201 consecutive colorectal carcinoma patients who were treated surgically in Chinese People’s Liberation Army General Hospital (Beijing, China) between August 1999 and September 2003 and 60 cases with distal normal mucosa were recruited in this study. There were 127 men and 74 women with ages ranging from 20 to 81 years (median, 62 years; mean, 57.8 years). The carcinomas were located in the cecum (n = 9), ascending colon (n = 29), transverse colon (n = 14), descending colon (n = 9), sigmoid colon (n = 48) and rectum (n = 92). Of these patients, 24 were grade I, 110 grade II and 67 grade III, according to histological grading; and 48 were stage I, 72 were stage II, 70 were stage III and 11 were stage IV, according to clinical TNM stage revised by International Union Against Cancer in 2003. All the patients were followed up for survival. During the follow-up period from the date of surgery until April 30, 2009, 126 patients died and 75 were alive (median survival time, 51 mo, range, 0.10-115 mo).

The resected specimens were fixed in 10% formalin. The gross appearance and the size of tumor were recorded. Patients’ medical records and histopathology of each specimen were reviewed. The tissues (cancer and normal) were cut into 5-mm slices, embedded in paraffin for histological and immunohistochemical examinations. Each paraffin block was cut at 4 μmol/L in thickness, and sections were mounted onto adhesive-coated slides. Slides were deparaffinized in xylene twice for 10 min and rehydrated through descending concentration of ethanol. Antigen retrieval was performed in 0.01 mol/L citrate buffer (pH 6.0) by microwave oven for 2 min and 30 s at 100°C. Endogenous peroxidase activity was blocked with 0.3% hydrogen peroxidase for 15 min. After washing with phosphate-buffered saline (PBS), the sections with primary polyclonal rabbit antibody to human LRP16 (recognized and isolated in 1999 by Department of Molecular Biology of our hospital) that was diluted 1:1000 in blocking solution were incubated at 4°C overnight, in a humidified chamber. After washing three times with PBS, the sections incubated for 30 min with biotinylated secondary antibody (polyperoxidase-anti-mouse/rabbit IgG, Zymed) then washed again with PBS. 3,3’-Diaminobenzidine was used as the chromogen. Sections were then counterstained with hematoxylin for 1 min, raised in water, dehydrated in ascending concentrations of ethanol followed by clearance with xylene, and cover slipped permanently for light microscopy. Normal ovary tissue was used as a positive control, whereas the primary antibody was replaced by PBS as a negative control.

Immunohistochemically stained slides were reviewed by two investigators independently who were blinded to all clinical data. Slides were analyzed under light microscopy using manual methods. Staining was graded for intensity of staining (0, negative; 1, weak; 2, moderate; 3, strong) and percentage of cells stained (0, < 5%; 1, 6%-25%; 2, 26%-50%; 3, > 50%). The final score was determined by the combined staining score (extent + intensity). Score 0 was defined as negative expression (-), scores 1-3 as weak staining pattern (+), and 4-6 as strong expression (++).

Statistical analysis was performed using SPSS 13.0 software (SPSS Inc., Chicago, Illinois). The χ² test was used to examine the various clinicopathological characteristics and LRP16 expression. Cumulative survival curves were drawn by the Kaplan-Meier method. The difference between the curves was analyzed by the Log-rank test. Multivariate survival analysis was based on Cox proportional hazard model. P < 0.05 was considered statistically significant.

LRP16 staining was performed in 201 colorectal cancer patients and 60 cases of normal tissues by IHC.The result showed that in cancer tissues, LRP16 staining was negative (-) in 41.8% (84/201) cases, weak positive (+) in 21.4% (43/201) cases and strong positive (++) in 36.8% (74/201) cases; whereas in normal mucosas, LRP16 was negative in 65% (39/60) cases, weak positive in 13.3% (8/60) and strong positive in 21.7% (13/60) cases. As a whole, LRP16 expression was detected in 58.2% (117/201) colorectal carcinoma, and in 35% (21/60) distal normal mucosa (Figures 1 and 2). The expression of LRP16 protein was found in cell nucleus or both cell nucleus and cytoplasm. The difference of LRP16 expression between colorectal cancer and normal mucosa was statistically significant (χ² = 9.998, P = 0.002).

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Figure 1 Positive leukemia related protein 16 (LRP16) expression in colorectal adenocarcinoma. The final score was 6, including 3 scores (strong) related to intensity of staining and 3 scores (> 50%) related to extent of staining. Thus it was defined as strong staining pattern (++). A: Immunohistochemistry (IHC), × 100; B: IHC, × 400.

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Figure 2 Negative LRP16 expression in normal colorectal mucosa (IHC, × 400). The final score was 0, including 0 score (negative) related to intensity of staining, and 0 scores (< 5%) related to extent of staining. Thus it was defined as negative expression (-).

Upon clinicopathological analysis, more LRP16 expression was present in bigger tumors (P = 0.001). When comparing the LRP16 status with clinicopathological variables, we found significant positive correlations between LRP16 expression and degree of differentiation (P = 0.010), depth of invasion (P = 0.001), lymph node metastasis (P = 0.001) and distant metastasis (P = 0.027). The level of LRP16 in cases of low TNM stage (I + II) was lower than that of high stage (III + IV) (χ² = 27.088, P = 0.001) (Table 1).

All 201 patients were followed up for survival to assess LRP16 expression as a prognostic factor. Kaplan-Meier survival curves and Log-rank test demonstrated that LRP16 positive cases showed a significantly shortened median survival time (38.0 mo) in comparison with other patients (105.0 mo) (Log rank = 41.455, P = 0.001) (Figure 3). The overall 5-year survival rate of the LRP16 negative patients (71.4%) was higher than that of the LRP16 positive group (29.1%). LRP16 expression appeared as a significant independent prognostic factor (P = 0.001) with a relative risk of 2.082 (confidence interval, 1.3-3.2) in the multivariate survival analysis of the patients. Other independent prognostic factors in multivariate survival analysis included tumor shape, degree of differentiation, depth of invasion and distant metastasis (Table 2).

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Figure 3 Cumulative survival according to LRP16 expression in colorectal cancer patients. The Kaplan-Meier plot of colorectal carcinoma patients (n = 201) demonstrates a significantly lower survival with LRP16 positive expression, P = 0.001.

Colorectal cancer is a major cause of cancer–related morbidity and mortality. In the Western world it is the second leading cause of death from cancer. The evidence of colorectal cancer has increased recently in China. Although this disease is curable by surgical interventions together with chemotherapy and radiation in its early stages, the patients are often asymptomatic before the metastasis occurred. Therefore, effective screening and preventive strategies for colorectal cancer are necessary to enhance our capability to predict the clinical outcome of the disease.

Leukemia related protein 16 (LRP16) gene plays an important role in the carcinogenesis and progression of hormone-dependent breast cancer. LRP16 expression was also reported to be associated with invasion, metastasis and prognosis of gastric carcinoma. But no comprehensive description of LRP16 protein expression in colorectal cancer has been reported. In this study, the authors investigated the expression patterns of LRP16 protein in human colorectal cancers and compared the clinical and pathological variables including survival time of the patients.

To date, only limited data on LRP16 protein expression in solid tumors are available. This study is believed to be the first trial for verifying the relationship between LRP16 expression and clinicopathological factors of colorectal carcinoma.

The authors presented some evidences to show a significant association between high LRP16 expression in colorectal cancer and early disease progression or the disease related death, and they believe that the LRP16 expression status detected by immunohistochemistry (IHC) may be a molecular marker to predict the prognosis of colorectal carcinoma patients.

In this paper the authors have analyzed by IHC the expression of LRP16 protein in 201 cases of colorectal carcinoma and 60 cases of distal normal mucosae and they have correlated the expression of this marker with the degree of differentiation, invasiveness, metastasis and prognosis of colorectal carcinoma. The study is well performed and the results are quite interesting, and are potentially helpful for clinical application, the quality of the research and presentation, including photomicrographs and figures, is high, the discussion is clear and well written.