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High Expression of Macrophage Colony-Stimulating Factor in Peritumoral Liver Tissue Is Associated With Poor Survival After Curative Resection of Hepatocellular Carcinoma

Xiao-Dong Zhu, Ju-Bo Zhang, Peng-Yuan Zhuang, Hong-Guang Zhu, Wei Zhang, Yu-Quan Xiong, Wei-Zhong Wu, Lu Wang, Zhao-You Tang, Hui-Chuan Sun

From the Liver Cancer Institute and Zhongshan Hospital, and Department of Pathology and Pathology Research Center, Shanghai Medical College, Fudan University, Shanghai, People's Republic of China

Corresponding author: Hui-Chuan Sun, MD, PhD, Liver Cancer Institute and Zhongshan Hospital, Fudan University, Shanghai 200032, China; e-mail: sun.huichuan{at}zs-hospital.sh.cn

	ABSTRACT

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION AUTHORS' DISCLOSURES OF... AUTHOR CONTRIBUTIONS Glossary Terms Appendix REFERENCES

 
Purpose To investigate prognostic values of the intratumoral and peritumoral expression of macrophage colony-stimulating factors (M-CSF) in hepatocellular carcinoma (HCC) patients after curative resection.

Patients and Methods Expression of M-CSF and density of macrophages (M) were assessed by immunohistochemistry in tissue microarrays containing paired tumor and peritumoral liver tissue from 105 patients who had undergone hepatectomy for histologically proven HCC. Prognostic value of these and other clinicopathologic factors was evaluated.

Results Neither intratumoral M-CSF nor M density was associated with overall survival (OS) or disease-free survival (DFS). High peritumoral M-CSF and M density, which correlated with large tumor size, presence of intrahepatic metastasis, and high TNM stage, were independent prognostic factors for both OS (P = .001 and P < .001, respectively) and DFS (P = .001 and P = .003, respectively) and affected incidence of early recurrence. In a small HCC subset, peritumoral M-CSF was also correlated with both OS and DFS (P = .038 and P = .001, respectively). The combination of peritumoral M-CSF and M had a better power to predict the patients' death and disease recurrence (P < .001 for both).

Conclusion High peritumoral M-CSF and M were associated with HCC progression, disease recurrence, and poor survival after hepatectomy, highlighting the importance of peritumoral tissue in the recurrence and metastasis of HCC. M-CSF and M may be targets of postoperative adjuvant therapy.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION AUTHORS' DISCLOSURES OF... AUTHOR CONTRIBUTIONS Glossary Terms Appendix REFERENCES

 
Hepatocellular carcinoma (HCC) is the sixth most common cancer and the third most common cause of death from cancer worldwide.¹ Although hepatectomy is the best method to provide long-term survival for patients with HCC,² high postoperative recurrence rate is a major problem. Biomarkers, mainly from tumor tissue or tumor cells, have been extensively studied,³ but so far, the results have not been satisfying. Metastasis or recurrence of HCC is mainly intrahepatic, which indicates that the peritumoral liver tissue may be a favorable soil for the spreading hepatoma cells. Budhu et al⁴ found that intrahepatic venous metastasis was associated with a unique immune/inflammation response signature in the peritumoral liver tissue but not in the intratumoral microenvironment, highlighting the influence that the peritumoral liver tissue has on prognosis and intrahepatic micrometastasis.

Macrophages (M) are a major component of the infiltrate of most tumors,⁵ which are attracted by chemokines, such as macrophage colony-stimulating factors (M-CSF), mainly produced by tumor cells.⁶ When appropriately activated, M can kill tumor cells or elicit tissue-destructive reactions.⁷ Recent evidence shows that M-CSF can also induce monocytes to produce more Th2 cytokines and fewer Th1 cytokines, after which the M present the so-called M2 phenotype^4,8,9 and secrete some growth factors that are essential for tumor growth and invasion.¹⁰ Inhibition of M-CSF could significantly decrease the infiltration of M, resulting in a significant delay in tumor progression; in contrast, by overexpression of M-CSF or supplementation of recombinant M-CSF, M recruitment was enhanced and correlated with accelerated tumor growth, progression, and angiogenesis.^11-18 Although the role of peritumoral M-CSF and M in intrahepatic metastasis has been revealed by Budhu et al,⁴ its prognostic value remains to be elucidated. Furthermore, the distribution of M-CSF and M in the peritumoral liver tissue is still not clear.

On the basis of this information, we hypothesized that high M-CSF expression in peritumoral liver tissue could recruit more M and facilitate the growth of a subclinical metastatic niche, increase the recurrence rate, and result in a dismal survival rate. Furthermore, we also intended to investigate the distribution of M-CSF by comparing the density of M-CSF expressed in peritumoral liver tissue at different distances from the tumor margin.

	PATIENTS AND METHODS

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Patients and Specimens
From January 1999 to March 2006, the same surgical team in our institute performed curative resection for HCC on 968 consecutive patients, defined as macroscopically complete removal of the tumor. The criteria for resectability have been described previously.¹⁹ One hundred five patients were randomly retrieved from a prospectively collected database. None of the patients received any preoperative anticancer treatment. Of them, 90 patients had hepatitis B history, and preoperative liver function was all classified as Child-Pugh class A. Tumor stage was determined according to the 2002 International Union Against Cancer TNM classification system.²⁰ Tumor differentiation was graded by the Edmondson grading system. The Scheuer system was applied in 100 patients (the surrounding liver tissue was not adequate to score in five patients) for grading (necroinflammatory activity) and staging (fibrosis and cirrhosis) of the nontumor liver tissue.^21,22 See detailed clinicopathologic features in Appendix Table A1 (online only). The study was approved by the Zhongshan Hospital Research Ethics Committee. Informed consent was obtained according to the committee's regulations.

Follow-Up and Postoperative Treatment
All patients were observed until March 2007, with a median observation time of 22.1 months. Follow-up procedures were described in our previous study.^19,23 Treatment modalities after relapse were administered according to a uniform guideline as previously described.^19,24,25 Overall survival (OS) was defined as the interval between the dates of surgery and death. Disease-free survival (DFS) was defined as the interval between the dates of surgery and recurrence; if recurrence was not diagnosed, patients were censored on the date of death or the last follow-up.

Tissue Microarray and Immunohistochemistry
A tissue microarray (TMA) was constructed as described previously.²³ Briefly, two cores were taken from each representative tumor tissue and from liver tissue adjacent to the tumor within a distance of 10 mm to construct TMA slides (in collaboration with Shanghai Biochip Company Ltd, Shanghai, China). Duplicate cylinders from two different areas, intratumoral and peritumoral (a total of four punches for each patient), were obtained. Then, two TMA sections with 105 pairs of tumors and matched peritumoral samples were constructed.

The immunohistochemistry protocols are described elsewhere²³ and in the Appendix (online only). Primary antibodies were mouse antihuman monoclonal antibodies combined with M-CSF (1:200; Santa Cruz Biotechnology, Santa Cruz, CA) and CD68 (1:100; Zymed Laboratories, San Francisco, CA). The components of the Envision-plus detection system (EnVision+/HRP/Mo; Dako, Carpinteria, CA) were applied. Reaction products were visualized by incubation with 3,3'-diaminobenzidine. Negative controls were treated identically but with the primary antibodies omitted (Appendix Figure A1, online only).

Evaluation of Immunohistochemical Findings
The density of positive staining was measured with the use of a computerized image system composed of a Leica CCD camera DFC420 connected to a Leica DM IRE2 microscope (Leica Microsystems Imaging Solutions Ltd, Cambridge, United Kingdom). Under high-power magnification (x200), photographs of four representative fields were captured by the Leica QWin Plus v3 software; identical settings were used for each photograph (see Appendix for details). The M-CSF density was counted by Image-Pro Plus v6.2 software (Media Cybernetics Inc, Bethesda, MD). For the reading of each antibody staining, a uniform setting for all the slides was applied. Integrated optical density of all the positive staining of M-CSF in each photograph was measured, and its ratio to total area of each photograph was calculated as M-CSF density (Appendix Figs A2 and A3, online only). CD68-positive areas in the photographs were measured by the Leica Qwin Plus, and the M density in each photograph was calculated as CD68-positive area/total area.

Long-Distance Peritumoral Sections
The other 18 specimens, which contained long-distance peritumoral tissue (at a distance of at least 30 mm from the tumor edge), were collected from patients who underwent hepatectomy for HCC and were immunostained with M-CSF and CD68 antibodies. Peritumoral tissues at three distances (5, 15, and 30 mm) from the tumor margin were observed, photographs of three hot spots (x200) at each distance were taken, and M-CSF and M densities were evaluated by the computerized imaging system described earlier. Univariate analysis of variance was performed with SPSS 13.0 for Windows (SPSS Inc, Chicago, IL) to reveal the distributions of M-CSF and M in peritumoral liver tissue. Paired-sample t test was applied to compare the densities between subsets.

Statistical Analysis
Analysis was performed with SPSS 13.0 for Windows (SPSS Inc); the Pearson ² test or Fisher's exact test was used to compare qualitative variables; and quantitative variables were analyzed by the t test or Pearson's correlation test. Kaplan-Meier analysis was used to determine the survival. Log-rank test was used to compare patients' survival between subgroups; the Cox regression model was used to perform multivariate analysis. Receiver operating characteristic (ROC) curve analysis was used to determine the predictive value of the parameters. P < .05 was considered statistically significant.

For M-CSF density, the cutoff for the definition of subgroups was the median value. For M density, a minimum P value was sought,^26,27 and the 75th percentile value (peritumoral M density = 5%; intratumoral M density = 3.7%) was defined as the cutoff value for high and low M density in this study (Appendix Fig A4, online only).

	RESULTS

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Immunohistochemical Findings in TMA
M-CSF staining was mainly on the cytoplasm of tumor cells or hepatocytes. Most of the stroma cells were negative staining, although sporadic positive staining on these cells was also observed (Figs 1A to 1D, Appendix Fig A2). The average levels of M-CSF and CD68 staining (Figs 1E to 1H) are shown in Table 1. Cores of two patients were unexpectedly detached from TMA sections during peritumoral M-CSF immunostaining. Peritumoral M-CSF and M density positively correlated with the densities in tumor tissue (r = 0.292, P = .003; and r = 0.234, P = .016, respectively; Fig 1) and were significantly higher than the densities in tumor tissue (P < .001 for both).

View larger version (88K): [in this window] [in a new window] [PowerPoint Slide for Teaching]   Fig 1. Photographs of immunostaining of (A to D) macrophage colony-stimulating factor (M-CSF) and (E to J) CD68 in tissue microarrays were taken for further analyses. Patient 23 (A, C, E, and G) showed high density of staining, whereas patient 65 (B, D, F, and H) showed low density (bar, 50 µm). (I) M-CSF and (J) macrophage (M) densities differed at different distances from tumor margin in long-distance peritumoral sections. (*) Paired samples t test showed a statistical difference between the two groups.

M-CSF and M Distribution in Peritumoral Liver Tissue

Correlations Between M-CSF/M Density and Clinicopathologic Features
As shown in Table 2, neither intratumoral M-CSF nor M density correlated with any clinicopathologic feature. However, patients with high peritumoral M-CSF or M density were prone to have large tumor size, high TNM stage, and high rates of intrahepatic metastasis. Average peritumoral M-CSF and M densities did not differ statistically between patients with high (grade 3 to 4; n = 42) and low (grade 1 to 2; n = 58) necroinflammatory activity in the peritumoral liver tissue (P = .292 and P = .618, respectively). Patients with cirrhosis (stage 4; n = 38) had significantly lower peritumoral M density than patients without definite cirrhosis (stage 1 to 3; n = 62; 3.00% v 4.34%, respectively; P = .001); average peritumoral M-CSF density did not differ statistically between these two groups (0.041 v 0.045, respectively; P = .185).

View this table: [in this window] [in a new window]   Table 2. Relationship Between Intratumoral and Peritumoral M/M-CSF and Clinicopathologic Features

View larger version (36K): [in this window] [in a new window] [PowerPoint Slide for Teaching]   Fig 2. Cumulative overall and disease-free survival curves of patients with high or low peritumoral features. (A and B) Low peritumoral macrophage colony-stimulating factor (M-CSF) and (E and F) low macrophage (M) density in peritumor were associated with prolonged overall and disease-free survival. (C, D, G, and H) The intratumoral features were associated with neither overall survival nor disease-free survival.

Using 12 months as the cutoff value, all of the recurrences were divided into early recurrence, which is mainly from intrahepatic metastasis, and late recurrence, which is usually a result of a multicentric new tumor.²⁸ More patients with high peritumoral M-CSF or M, compared with patients with low peritumoral M-CSF or M, had an early recurrence (26 of 51 v five of 52 patients, respectively; P < .001; 12 of 26 v 20 of 79 patients, respectively; P = .045) rather than a late recurrence (four of 51 v five of 52 patients, respectively; P = .99; four of 26 v five of 79, respectively; P = .220). To eliminate the impact of tumor size on the patients' survival, we further investigated the prognostic factors in the subgroup with small HCC (maximum diameter 5 cm, n = 57). The peritumoral M-CSF and hepatitis B e antigen correlated with both OS (P = .038 and P = .008, respectively) and DFS (P = .001 and P = .015, respectively) in this subgroup. Other factors, including intratumoral M-CSF/M and peritumoral M, did not correlate with OS or DFS.

Combination of Peritumoral M-CSF and M Density and ROC Analysis
Patients were classified into four groups according to their peritumoral M-CSF and M densities: group I (n = 44), low M-CSF and M density; group II (n = 33), high M-CSF but low M density; group III (n = 8), low M-CSF but high M density; and group IV (n = 18), high M-CSF and M density. Differences in both OS (P < .001) and DFS (P < .001) were significant among the four groups. The 5-year OS and DFS rates were 80% and 67%, respectively, for group I but only 11% and 0%, respectively, for group IV (Figs 3A and 3B).

View larger version (23K): [in this window] [in a new window] [PowerPoint Slide for Teaching]   Fig 3. Cumulative (A) overall and (B) disease-free survival curves of the combination of peritumoral macrophage colony-stimulating factor (M-CSF) and macrophages (M; see Results for details). (C and D) All factors adopted in receiver operating characteristic analysis predicted death and recurrence precisely during follow-up (P < .05 for all). The predictive value of this combination was the best one (see Appendix Table A2, online only). FPF, false-positive fraction; TPF, true-positive fraction; UICC, International Union Against Cancer.

	DISCUSSION

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In the present study, we found that a high density of M-CSF and M in the peritumoral liver tissue, but not in tumor tissue, was associated with a high incidence of intrahepatic metastasis and poor survival after resection of primary tumor. The combination of peritumoral M-CSF and M density had a better power to predict patients' outcomes. Therefore, we propose that the peritumoral inflammation/immune environment is important in understanding the mechanism of intrahepatic metastasis of HCC and in shaping the postoperative strategy for prevention of recurrence after hepatectomy.

The counts of M were higher in peritumoral liver tissue than in tumor tissue, as reported previously by others.^29,30 Budhu et al⁴ found that M-CSF staining is mainly confined to peritumoral hepatocytes, which can be detected more frequently in HCC samples with intrahepatic venous metastasis. The present study supports this finding. Overexpression of M-CSF in a tumor is common in many types of cancer and correlates with poor survival.^31-36 However, the mechanism of overexpression of M-CSF and higher density of M in the peritumoral liver tissue remains unclear. In the present study, we found a parallel M-CSF expression level in the tumor and peritumoral hepatocytes, implying that the baseline level of M-CSF expression differs among patients. We found that the expression level of intratumoral or peritumoral M-CSF did not correlate with any parameters related to hepatitis, including serum ALT, presence of hepatitis B e antigen, and necroinflammation activity and cirrhosis scores. Therefore, it could be possible that the baseline expression level of M-CSF is associated with some other genetic heterogeneity among different individuals. However, the centripetal distribution of M-CSF expression in peritumoral hepatocytes implied that the tumor is a stimulator. This distribution could be caused by hypoxia in hepatocytes resulting from compression by the primary tumor, especially in patients with large tumors. It has been reported that placenta^37,38 and endothelial cells³⁹ could excrete more M-CSF in a hypoxic environment. Another possibility, as described by Dvorak⁴⁰ 20 years ago, is that tumors are wounds that do not heal. Our version of this hypothesis is that a tumor itself is also a stimulator of chronic inflammation and causes a reaction in host tissue (hepatocytes), which produces a number of cytokines and attracts M at the tumor-host margin. Therefore, the expression level of M-CSF in peritumoral liver tissue depends on the baseline expression that may be associated with the genetic background or other inflammation factor evenly distributed in the liver in addition to the elevated expression of M-CSF that is stimulated by the tumor. This hypothesis is supported by the elegant study that showed that an implanted or spontaneous tumor led to accumulation of vascular endothelial growth factor around the tumor, which was much stronger than its accumulation inside the tumor,⁴¹ which is similar to our observation.

In the present study, we found that the density of M-CSF and M in the peritumoral liver tissue, but not in tumor tissue, was associated with patients' survival, even in a subgroup of patients with small HCC. We think that intratumoral and peritumoral microenvironments play different roles in the progression of HCC. Intratumoral M-CSF and M2-M may be involved in facilitating the dissemination of the tumor cells, but this step is not a rate-limiting step in metastasis.^42-44 However, in the peritumoral liver tissues, M-CSF and M may be involved in facilitating colonization and growth of the micrometastasis, which is always a rate-limiting step.⁴² Furthermore, the impact of intratumoral M-CSF and M was removed by surgery. Therefore, as the front line of defense to prevent tumor growth, the peritumoral liver tissue, which is endowed with abundant M-CSF and M,⁴¹ plays an opposite role by providing a fertilized soil for subclinical metastatic tumor cells. Recently, the role of M in preparing soil in premetastasis niche has also been reported.⁴⁵ The mechanism of peritumoral M involved in intrahepatic metastasis of HCC could be similar.

High incidence of intrahepatic metastasis and recurrence after resection suggested that peritumoral environment is an important but often neglected issue. Few studies that focus on peritumoral tissue have been reported. Ezaki et al^46,47 found that higher peritumoral expression of thymidine phosphorylase was associated with a higher incidence of postoperative recurrence. Yu et al⁴⁸ found that blood vessel density was higher in peritumoral tissue compared with blood vessel density in tumor, which was consistent with elevated VEGF₁₆₅ and HIF-1 expression in peritumoral hepatocytes. Budhu et al⁴ found that a peritumoral expression signature could predict vascular invasion. Okamoto et al⁴⁹ found that a specific gene profile in noncancerous liver tissue could predict multicentric occurrence or recurrence of HCC. Together with these results, the present study implies that postoperative adjuvant therapies should target not only the residual tumor cells, but also the soil to make it resistant to tumor growth. Obviously, M-CSF and M could also be good targets for adjuvant therapy after hepatectomy.

On the whole, the present study indicates that M-CSF expressed by peritumoral liver cells can predict the postoperative survival of patients with HCC and also highlights the important role that the residual liver may play in recurrence and metastasis. One day, M-CSF and M may become new targets of adjuvant therapy; they await further investigation.

	AUTHORS' DISCLOSURES OF POTENTIAL CONFLICTS OF INTEREST

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION AUTHORS' DISCLOSURES OF... AUTHOR CONTRIBUTIONS Glossary Terms Appendix REFERENCES

 
The author(s) indicated no potential conflicts of interest.

	AUTHOR CONTRIBUTIONS

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION AUTHORS' DISCLOSURES OF... AUTHOR CONTRIBUTIONS Glossary Terms Appendix REFERENCES

 
Conception and design: Ju-Bo Zhang, Wei-Zhong Wu, Lu Wang, Zhao-You Tang, Hui-Chuan Sun

Administrative support: Zhao-You Tang

Provision of study materials or patients: Ju-Bo Zhang, Peng-Yuan Zhuang, Wei Zhang, Hui-Chuan Sun

Collection and assembly of data: Ju-Bo Zhang, Peng-Yuan Zhuang, Hong-Guang Zhu, Wei Zhang, Yu-Quan Xiong, Hui-Chuan Sun

Data analysis and interpretation: Xiao-Dong Zhu

Manuscript writing: Xiao-Dong Zhu, Hui-Chuan Sun

Final approval of manuscript: Xiao-Dong Zhu, Hui-Chuan Sun

	Glossary Terms

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Tissue microarray:

Used to analyze the expression of genes of interest simultaneously in multiple tissue samples, tissue microarrays consist of hundreds of individual tissue samples placed on slides ranging from 2 to 3 mm in diameter. Using conventional histochemical and molecular detection techniques, tissue microarrays are powerful tools to evaluate the expression of genes of interest in tissue samples. In cancer research, tissue microarrays are used to analyze the frequency of a molecular alteration in different tumor type, to evaluate prognostic markers, and to test potential diagnostic markers.

ROC (receiver operating characteristic) curves:

ROC curves plot the true positive rate (sensitivity) against the false-positive rate (1-specificity) for different cut-off levels of a test. The area under the curve is a measure of the accuracy of the test. An area of 1.0 represents a perfect test (all true positives), whereas an area of 0.5 represents a worthless test.

Immunohistochemistry:

The application of antigen-antibody interactions to histochemical techniques. Typically, a tissue section is mounted on a slide and is incubated with antibodies (polyclonal or monoclonal) specific to the antigen (primary reaction). The antigen-antibody signal is then amplified using a second antibody conjugated to a complex of peroxidase-antiperoxidase (PAP), avidin-biotin-peroxidase (ABC) or avidin-biotin alkaline phosphatase. In the presence of substrate and chromogen, the enzyme forms a colored deposit at the sites of antibody-antigen binding. Immunofluorescence is an alternate approach to visualize antigens. In this technique, the primary antigen-antibody signal is amplified using a second antibody conjugated to a fluorochrome. On UV light absorption, the fluorochrome emits its own light at a longer wavelength (fluorescence), thus allowing localization of antibody-antigen complexes.

M-CSF (macrophage colony-stimulating factor):

A secreted cytokine synthesized by mesenchymal cells. By binding to its receptors, M-CSF stimulates the survival, proliferation, and differentiation of hematopoietic cells of the monocyte-macrophage lineage. It may also be involved in development of the placenta.

Macrophages (M):

Macrophages which originate from monocytes belong to the innate immune system. Main types are peritoneal macrophages; alveolar macrophages; histiocytes; Kupffer cells of the liver; and osteoclasts. They act in non-specific defense (or innate immunity) as well as specific defense (or cell-mediated immunity) of vertebrate animals. Their role is to phagocytose cellular debris and pathogens either as stationary or mobile cells, and to stimulate lymphocytes and other immune cells to respond to the pathogen.

M2 phenotype (type II macrophages, M2 polarized macrophages):

Under the stimulation of specific cytokines, such as IL-4, IL-10, IL-13, and M-CSF but not GM-CSF, LPS, INF-, etc., macrophages tune inflammation and adaptive immunity, promote cell proliferation by producing growth factors and products of the arginase pathway, scavenge debris by expressing scavenger receptors, and promote angiogenesis and tissue remodeling and repair, suppress the adaptive immunity, and promote tumor.

Scheuer system:

The scoring system was described originally for chronic viral hepatitis, it is now applied to non-viral hepatitis as well. It gives the portal and lobular components of activity equal weight and evaluates both the necroinflammatory activity and liver fibrosis/cirrhosis in chronic hepatitis.

IOD (integrated optical density):

A computer-assisted method usually used to quantify both the area and the intensity of the positive staining of immunohistochemistry.

Microvascular invasion:

The presence of tumor cells inside the lumen of the microvasculature, including minor vascular invasion and microscopic vascular invasion, was defined as microvascular invasion. It is different from major vascular invasion which refers to gross invasion of the right or left main branches of the portal or hepatic veins.

	Appendix

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Immunohistochemistry Protocols
The sections were dewaxed in xylene and graded alcohols, hydrated, and washed in phosphate-buffered saline. After the endogenous peroxidase was inhibited by 3% H₂O₂ for 30 minutes, the sections were pretreated in a microwave oven (14 minutes in sodium citrate buffer; pH = 6) and then incubated with 10% normal goat serum for 30 minutes. Primary antibodies composed of mouse antihuman monoclonal antibodies combined with macrophage colony-stimulating factor (M-CSF) (1:200; Santa Cruz Biotechnology, Santa Cruz, CA) and CD68 (1:100; Zymed Laboratories, San Francisco, CA) were applied overnight in a moist chamber at 4°C. After the primary antibody was washed off, the components of the Envision-plus detection system were applied with an antimouse polymer (EnVision+/HRP/Mo; Dako, Carpinteria, CA). Reaction products were visualized by incubation with 3,3'-diaminobenzidine and then counterstained with hematoxylin. Negative controls were treated identically but with the primary antibody omitted.

Settings for Macrophages/M-CSF Immunostaining Evaluations
Capture settings of the computerized image system were as follows: (1) configure of Leica CCD camera DFC420 (Leica Microsystems Imaging Solutions, Ltd, Cambridge, United Kingdom): voltage of light source: 6.9V; diaphragm of a camera: BF: 8; (2) configure of Leica QWin Plus v3 software (Leica Microsystems Imaging Solutions): exposure time: 15.6 mses; gain: 1.0x; saturation: 1.5; and (3) stored in computer in tiff format.

Ratio of the area of stained cells (such as endothelial cells, macrophages [M], and smooth muscle cells) (Fischer C, Jonckx B, Mazzone M, et al. Cell 131:463-475, 2007; Wild R, Ramakrishnan S, Sedgewick J, et al. Microvasc Res 59:368-376, 2000) to the total area of the field was an established method to grade the positive staining and avoided the subjectivity of manual grading. In this study, we used the same method to count the number of M.

However, M-CSF expression was diffused in both peritumor and intratumor area; therefore, intensity of staining also needed to be measured when evaluating M-CSF expression. Integrated optical density (IOD) evaluates both the area and intensity of the positive staining (Hayat MA. Quantitation of immunostaining, Microscopy, Immunohistochemistry, and Antigen Retrieval Methods for Light and Electron Microscopy. New York, NY, Springer, 2002, pp 105-108). We calculated the IOD of each photograph acquired from the tissue microarray sections by using Image-Pro Plus v6.2 software (Media Cybernetics Inc, Bethesda, MD). Briefly, the typical positive staining area was located with the help of a pathologist (H.-G.Z.) in the Segmentation panel; Standard Optical Density was chosen in the Intense Calibration panel, and background was subtracted in the panel of Optical Density Calibration. IOD values of all the areas in each photograph were exported into an Excel document for further analyses. The mean IOD (IOD/total area) represents the M-CSF expression level of in peritumor or intratumor area. See Appendix Figure A2, which shows how this method was conducted.

Configuration of color of interest for all CD68 staining in Leica QWin Plus (Leica Microsystems Imaging Solutions) was as follows: R, 173-34; G: 129-16; and B, 80-0. Configuration of color of interest for M-CSF staining in Image-Pro Plus v6.2 software (Media Cybernetics) was single for each feature, including peritumoral/intratumoral tissue microarrays and long-distance peritumoral sections staining.

View larger version (98K): [in this window] [in a new window] [PowerPoint Slide for Teaching]   Fig A1. Negative controls of immunohistochemistry staining in (A) peritumoral liver tissue and (B) intratumoral tissue in this study showed almost no nonspecific staining.

View larger version (80K): [in this window] [in a new window] [PowerPoint Slide for Teaching]   Fig A2. Representative photographs of computer-assisted macrophage colony-stimulating factor (M-CSF) staining evaluation. Taking Figure 1A as an example, (A) is the original photograph acquired from tissue microarray sections (x200). Black arrows indicate the week positive staining stroma cells, and blue arrows indicate the negative staining stroma cells. The small rectangle of white area was set as the background noise. (B) The positive staining cytoplasm that was chosen as the color of interest and masked with red color, including the black arrowed stroma cells, but not the blue arrowed ones. Integrated optical density (IOD) of positively staining area was counted by computer, and the mean IOD represents the expression level of M-CSF by this patient in his/her peritumoral liver tissue. (C) The unchosen area, which is masked with black color.

View larger version (13K): [in this window] [in a new window] [PowerPoint Slide for Teaching]   Fig A3. Scatter plots show that computer-assisted integrated optical density (IOD) evaluation correlated well with manual evaluation of (A) peritumoral and (B) intratumoral macrophage colony-stimulating factor (M-CSF) staining. (*) Spearman's correlation analyses; bars, 95% CI; –, negative staining; +, weak positive; ++, moderate positive; +++, strong positive.

View larger version (17K): [in this window] [in a new window] [PowerPoint Slide for Teaching]   Fig A4. Minimum P value seek was conducted in log-rank survival analysis when using different percentile values as cutoffs (Galon J, Costes A, Sanchez-Cabo F, et al. Science 313:1960-1964, 2006; Toi M, Bando H, Ogawa T, et al. Int J Cancer 98:14-18, 2002). When using the 60th, 65th, 70th, 75th, and 80th percentile levels of peritumoral macrophage (M) density as cutoff values, we could discriminate the disease-free survival (DFS), and the 75th percentile value (peritumoral M density = 0.05%) was the best one among them. P values were significant within a long range of cutoff values (from 60th to 80th percentiles), which suggested a good reproducibility. However, no cutoff level in intratumoral M density could discriminate patients' survival.

View larger version (89K): [in this window] [in a new window] [PowerPoint Slide for Teaching]   Fig A5. Consecutive sections of a representative long-distance peritumoral specimen (patient 3) were stained with (A to C) macrophage colony-stimulating factor (M-CSF) and (D to F) CD68. Both the M-CSF and macrophage (M) densities decreased with the distances further away from the tumor edge (bar, 50 µm; consecutiveness marked by black brush).

View larger version (31K): [in this window] [in a new window] [PowerPoint Slide for Teaching]   Fig A6. Schematic diagram of the distribution of (left) macrophage colony-stimulating factor (M-CSF) and (right) macrophages (M) of each patient (patients 1 to 18) in long-distance peritumoral sections. The color depth represents the relative density of M-CSF and M of each patient.

	NOTES

 
Supported by Grants No. 30672037 and 30300400 from the National Natural Science Foundation of China and the Foundation of China National "211" Project for Higher Education.

Both X.-D.Z. and J.-B.Z. contributed equally to this work.

Terms in blue are defined in the glossary, found at the end of this article and online at www.jco.org.

Authors' disclosures of potential conflicts of interest and author contributions are found at the end of this article.

	REFERENCES

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION AUTHORS' DISCLOSURES OF... AUTHOR CONTRIBUTIONS Glossary Terms Appendix REFERENCES

 
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Submitted December 9, 2007; accepted February 19, 2008.

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