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in Clinical and Experimental Therapeutics

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Volume 1 - 2001

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©2000-2005. All Rights Reserved. Therapeutic Solutions LLC

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Angiogenesis Does Not Correlate with Rectal Cancer Metastasis*


Mokenge P. Malafa, MDa

J. Mark Barnett, MDa

Theresa Karich, MDa

Leslie Neitzel, MSa

Brian Webb, MDb


aDepartment of Surgery

bDepartment of Pathology

Southern Illinois University School of Medicine

PO Box 19638

Springfield, IL 62794


Key Words: rectal cancer, metastasis, angiogenesis, factor VIII, survival



To determine the usefulness of angiogenesis in identifying rectal cancer patients with lymph node metastases, we compared the number of microvessels in the tumors of 22 patients with lymph node–positive rectal cancer to 22 patients with lymph node–negative rectal cancer. Patients were matched for tumor depth, histology, and grade. Microvessel counts in the tumors of patients with rectal cancer did not correlate with lymph node metastasis. Microvessel counts were 21.86 ± 12.99 for lymph node­–positive versus 19.14 ± 14.44 for lymph node–negative patients (P = .5138). Furthermore, the disease-free survival of patients with high microvessel counts was not significantly different from that of patients with low microvessel counts (P = .5103). Thus our data reject the hypothesis that angiogenesis is useful in identifying the presence of lymph node metastases in patients with rectal cancer. Additionally, in contrast to previous studies, patients with rectal cancer did not demonstrate an adverse impact of increased microvessel counts on disease-free survival.


*This study was supported by grants from the William E. McElroy Charitable Foundation and the Memorial Medical Center Foundation.



There is intense interest in identifying predictive features of lymph node metastasis in rectal cancer because this tumor is increasingly undergoing minimal surgical treatment.1-5 Local excision for rectal cancer may spare patients extensive resection and colostomy. One of the concerns of minimal therapies is that lymph nodes containing metastases may be left in the patient, thus resulting in treatment failure. Current clinical methods, such as radiologic assessment by endorectal ultrasound, have significant limitations with false- negative rates of 14.3% and an overall accuracy of only 63.5%.6,7 The pathologist is often called on to comment on histologic features of the primary tumor, which may predict aggressive behavior. Despite many documented adverse histologic features, there remains no accurate means of predicting metastatic behavior.8-11

Angiogenesis has been shown to be predictive of lymph node metastasis.12 Microvessel counts in tumors reflect the amount of angiogenesis within a tumor and have been proposed as useful predictors of lymph node metastasis. Previous work by others and ourselves has demonstrated that microvessel counts are predictive of lymph node metastasis in breast cancer and melanoma.13,14 There has been a great deal of interest in this finding as it relates to colorectal cancer. Several studies have reported mixed results regarding the relationship between angiogenesis and rectal cancer progression.15-20 These studies were not specifically designed to examine the impact of angiogenesis in rectal cancer lymph node metastasis.

In this study, we use a case control approach to examine the impact of angiogenesis on identifying rectal cancer patients with lymph node metastases. Our study examined the number of vessel counts in the tumors of 22 patients with lymph node­ positive rectal cancer compared with 22 patients with lymph nodenegative rectal cancer treated from 1984 to 1994.


Materials and Methods


Following approval by the Springfield Committee for Research Involving Human Subjects, the tumor registry at Memorial Medical Center in Springfield, IL, was used to obtain a database of patients who underwent surgery for rectal cancer between the years 1984 and 1994. Cases for which lymph node status, relevant clinical information, or paraffin blocks could not be obtained were excluded from the study. Ultimately, 22 patients with rectal cancer and positive lymph nodes and 22 patients with rectal cancer and negative lymph nodes were selected. Their respective paraffin-embedded tumor specimens were obtained. The two groups were matched for major risk factors for metastasis, including gender, age, tumor histology, T stage, tumor grade, and tumor size (Table 1). Information regarding T stage and lymph node status was obtained from the original surgical pathology reports. All other information was obtained from either the tumor registry or the patient’s medical record.



Formalin-fixed, paraffin-embedded tissue sections were heated at 60°C for 30 minutes, deparrafinized, and rehydrated through a graded series of alcohol and water. The sections were permeabilized with trypsin (1 mg/mL) for 5 minutes and rinsed thoroughly with water. To block endogenous peroxidase activity, sections were incubated with hydrogen peroxide diluted with phosphate-buffered saline (PBS) for 5 minutes and rinsed with PBS. Nonspecific binding of the secondary antibody was blocked with normal horse serum for 20 minutes. Sections were incubated with a 1:50 dilution of mouse antihuman factor VIII monoclonal antibody (DAKO Corp., Carpinteria, CA) for 30 minutes and rinsed with PBS. Immunoperoxidase staining was performed with an avidinbiotin complex method (VECTASTAIN Elite ABC kit, VECTOR Laboratories, Inc., Burlingame, CA) and DAB substrate (Research Genetics, Huntsville, AL). Rinsing with distilled water terminated the reaction. The sections were placed in a 0.1 M sodium acetate buffer for 10 minutes, counterstained with methyl green for 10 minutes, dehydrated, and mounted. Omission of the primary antibody was employed as a negative control, and reaction of the primary antibody with human uterine tissue was employed as a positive control.


Quantification for Tumor Vascularity

For each case, the rectal tumor specimen was scanned microscopically with an Olympus BH2 microscope to find the area of greatest vascular density. The number of microvessels per microscopic field (200х and 400х magnification) was counted for the area of greatest vascularity within the tumor. Structures were counted as microvessels if they stained positively for the endothelial cell marker, factor VIII, and appeared as an individual microvessel or endothelial cell cluster. Although lumens were generally present, this was not a criterion for a structure to be counted as a microvessel. Vessel counts were assessed without knowledge of patient outcome and were performed simultaneously by two independent investigators using a double-headed light microscope. Cases were also scored separately in a blinded fashion by a third investigator.



Independent group t-tests were used to compare the two patient groups (lymph node positive versus lymph node negative) on both the continuous and ordinal measures. Chi- squared tests of independence or Fisher’s exact test, when appropriate, were used to compare the two groups in regard to the categorical data. A logrank test was used to compare survival curves. Results are reported as mean ± standard deviation and are considered significant for P < .05.



Microvessel Counts Do Not Correlate with Lymph Node Metastasis in Rectal Cancer

Patients with lymph node metastases at the time of surgery had no statistically significant difference in number of microvessels compared with patients without lymph node metastases (Figure 1). Microvessel counts were 21.86 ± 12.99 versus 19.14 ± 14.44 (P = .5138) per 200х field and 11.55 ± 6.142 versus 9.864 ± 7.415 (P = .4255) per 400х field for lymph node–positive versus lymph node–negative groups, respectively. The median counts were 19.00 and 13.00 per 200х field and 10.00 and 6.50 per 400х field for lymph node–positive tumors versus lymph node–negative tumors, respectively.


Angiogenesis Is Not Predictive of Survival in Rectal Cancer

The median number of microvessel counts for all patients in the study was 18 at 200х magnification. To determine the effect of microvessel counts on disease-free survival, the patients were stratified into two groups, those with microvessel counts greater than or equal to 18, and those with microvessel counts less than 18. This stratification was made irrespective of a patient’s node status. Data was then plotted using patient’s survival status at last follow-up. There was no statistically significant difference in disease-free survival between patients with high versus low vessel counts (P = .5103; Figure 2). When patients were stratified by metastatic status and microvessel counts, angiogenesis still had no impact on disease-free survival (P = .4047 for node-positive patients) and (P = .1360 for node-negative patients; Figure 3).



Our study indicates no correlation between angiogenesis as measured by microvessel counts and lymph node metastasis in rectal cancer. The literature regarding colorectal cancer and angiogenesis reports conflicting results in terms of the impact of this process on lymph node metastasis.15-17,19,20 Our findings support the conclusions by Saclarides and coworkers16 and Vermeulen and coworkers20 who did not find a correlation between angiogenesis and lymph node metastasis. In contrast, our study refutes the findings of Takebayashi and coworkers who reported a correlation between microvessel counts and lymph node metastasis.19 It is important to note that the study by Takebayashi and colleagues did not differentiate between rectal and colon tumors but instead grouped them together as colorectal cancers. We feel that this grouping may obscure the unique biology of rectal cancers.

We specifically designed this study to determine the impact of angiogenesis on identifying patients with lymph node metastases. The strengths of our study include the focus on rectal tumors and the use of a control group matched for known factors, which may influence the metastatic phenotype (tumor depth, grade, histology). The weakness of our study is its retrospective nature and relatively modest number of subjects. Prospective studies with larger patient cohorts will be needed to confirm our findings.

Angiogenesis has been correlated with survival in patients with diverse malignancies, including rectal cancer.12,15,16,19,20 We did not observe a significant impact of angiogenesis on patient disease-free survival. This is in contrast to the results of other studies.15,16,19,20 Saclarides and colleagues noted a significant increase in vessel counts in patients who lived less than 5 years compared with those who lived more than 5 years.16 However, these patients were not controlled for risk factors associated with rectal cancer survival. Takebayashi and coworkers, Vermeulen and coworkers, and Ishikawa and coworkers used univariate and multivariate Cox proportional hazard regression models to demonstrate that microvessel counts were an independent prognostic factor in the survival of patients with rectal cancer.15,19,20 However, these studies have a limited value in determining prognosis in individual cases because there are overlaps and large standard deviations of microvessel counts related to the presence or absence of other pathologic or prognostic factors. Our observation of the absence of a correlation between microvessel counts and survival reflects our matched patient population. The use of microvessel counts to predict mortality in patients with rectal cancer should be evaluated in prospective controlled trials.



Angiogenesis is currently an important target for novel antitumor therapy and prognostication. We had hoped to uncover a predictive value of angiogenesis in rectal cancer lymph node metastasis in order to include this factor in selecting patients for local therapy. We discovered significant heterogeneity in the angiogenic phenotype of rectal tumors. Future clinical studies are needed to better define the role of angiogenesis for specific applications to patient care.



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Figure 1. (A) A representative photomicrograph showing immunostaining for factor VIII in rectal cancer. Many small microvessels positive for factor VIII were scattered in the tumor stroma. (B) Correlation between microvessel counts and lymph node metastasis. The number of microvessels in the rectal tumors of patients with lymph node metastases were not statistically different from the number in patients without lymph node metastases (P = .5138 per 200х field and P = .4255 per 400х field).


Figure 2. Prognostic significance of microvessel counts for disease-free survival in patients with rectal cancer regardless of lymph node status. Survival analysis for microvessel counts in relationship to the recurrence of cancer in a total of 44 patients was performed using a log rank test. A cutoff level of 18 microvessels was used because this was the median number of microvessels for all patients combined. There was no significant difference between the curves (P = .5103).


Figure 3. Prognostic significance of microvessel counts for disease-free survival in patients with (A) and without (B) lymph node metastases. There was no significant difference in disease-free survival for patients with (P = .4047) or without (P = .1360) lymph node metastases.


Table 1

Clinicopathologic Characteristics of 44 Patients with Rectal Cancer

            Lymph Node (positive)            Lymph Node (negative)            P Value

Number of patients 22            22            --

Age (years)            68.5 ± 9.989   70.05 ± 8.627            .5858

Gender (male/female)            12/10            11/11            1.0000

Tumor histology

  Adenocarcinoma          21            21            1.5116

  Mucinous            1            1            --

T stage (T2/T3)*            3/19            3/19            1.3360

Tumor grade (1/2/3)            3/15/4            8/11/3            .2197

Tumor size (cm)     4.891 ± 2.104            4.873 ± 1.240            .9723

Nodes examined            7.619 ± 5.590            6.810 ± 4.262            .6006

Chemotherapy            9/22            3/22            .0904

Radiation therapy 10/22            7/22            .4310

*T stage: tumor depth of invasion as defined by AJCC.

Tumor grade: 1 = poorly differentiated, 2 = moderately differentiated, 3 = well differentiated.