Correlative study of microvessel density and 5-lipoxygenase expression in human sporadic colorectal cancer.
Abstract: * Context.--5-Lipoxygenase (5-LO) is an arachidonic acidmetabolizing enzyme, which has been demonstrated to exert a role in colorectal cancer tumorigenesis. Its activity in promoting neoangiogenesis in colorectal malignancies has been also recently theorized on the basis of in vitro studies.

Objective.--To investigate whether any correlation existed between 5-LO immunoexpression amount and the quantity of neoangiogenesis, as reflected by microvessel density (MVD) in human sporadic surgically resected colorectal adenocarcinomas.

Design.--A total of 45 formalin-fixed, paraffin-embedded colorectal adenocarcinomas were submitted to the immunohistochemical procedures for 5-LO and CD105, which represent specific markers for neoangiogenesis and which were used in the assessment of MVD.

Results.--CD105-positive, intratumoral, newly formed vessels were present in 45 of 45 cases with variable MVD values. A 5-LO-positive immunohistochemical reaction was also found in 45 of 45 cases. A significantly higher MVD was evident in cases displaying a high 5-LO amount in comparison with those characterized by a low 5-LO expression (28.33 vs 19.44 vessels per [mm.sup.2]; P = .02). In addition, a positive significant correlation emerged between 5-LO immunoexpression amount and the MVD counts (r = 0.2986, P = .04).

Conclusions.--Our study demonstrates the existence of a relationship between 5-LO expression and the neoangiogenesis process as reflected by intratumoral MVD in human sporadic colorectal adenocarcinomas, thus suggesting that 5-LO may modulate the formation of blood vessels in these neoplasias.
Article Type: Report
Subject: Neovascularization (Research)
Colorectal cancer (Development and progression)
Capillaries (Properties)
Correlation (Statistics) (Methods)
Gene expression (Research)
Authors: Barresi, Valeria
Vitarelli, Enrica
Tuccari, Giovanni
Barresi, Gaetano
Pub Date: 11/01/2008
Publication: Name: Archives of Pathology & Laboratory Medicine Publisher: College of American Pathologists Audience: Academic; Professional Format: Magazine/Journal Subject: Health Copyright: COPYRIGHT 2008 College of American Pathologists ISSN: 1543-2165
Issue: Date: Nov, 2008 Source Volume: 132 Source Issue: 11
Topic: Event Code: 310 Science & research
Geographic: Geographic Scope: United States Geographic Code: 1USA United States
Accession Number: 230246871
Full Text: Arachidonic acid, a fatty acid mainly derived from dietary sources, is metabolized by 3 major pathways: (1) the cyclooxygenase pathway, which leads to the formation of prostaglandins (PGs); (2) the lipoxygenase (LO) pathway, which forms hydroperoxyeicosatetraenoic acids, hydroxyeicosatetraenoic acids, and leukotrienes; and (3) the cytochrome P-450 monooxygenase pathway, which leads to the formation of epoxydes. (1)

Three major LOs have been characterized in human tissues. They have been named 5-LO, 12-LO, and 15-LO for their ability to insert molecular oxygen at the 5, 12, or 15 carbon atom of arachidonic acid. These enzymes have been demonstrated to exert a protumorigenic role in human neoplasias. Indeed, the significant elevation of their metabolites has been evidenced in several malignancies, in accordance with their role in tumor growth, progression, and angiogenesis (2-4) and their involvement in various steps of carcinogenesis. (5, 6)

The 5-LO involvement in cancer pathobiology is indicated by its overexpression, together with one of its terminal products, LTB4, in a broad variety of cancer cells (4, 6-9) and human cancer tissues. (10-13) Specifically, higher 5-LO amounts have been detected in human colorectal cancer cells or tissues in comparison with the corresponding normal colonic cells or epithelium, (14-17) thus suggesting 5-LO's role in colorectal tumorigenesis. The hypothesis, according to which 5-LO contributes to colorectal carcinogenesis, has been further confirmed by the reduction of tumor growth obtained as a result of the use of 5-LO-specific inhibitors in in vitro cigarette smoke-induced colorectal cancer. (17) Besides the protumorigenic action, a proangiogenic role has been proposed for 5-LO in colorectal malignancies. Indeed, in vitro studies on cigarette smoke-induced colon cancer have also shown the effective inhibition of tumor angiogenesis as a consequence of 5-LO selective suppression. (17)

In the present study, we investigated the possible correlations between 5-LO expression amount and tumor neoangiogenesis in a series of human sporadic surgically resected colorectal adenocarcinomas. In particular, our purpose was to verify whether any difference in the quantity of neoangiogenesis was present between cases with a low and a high 5-LO expression, consistent with the hypothesis that angiogenesis is mediated through the increase in 5-LO expression in colorectal cancer. Indeed, if confirmed, this assumption might open new perspectives in the therapy of this neoplasia, with the 5-LO-immunoexpressing colorectal carcinomas eventually treated with both antiangiogenic drugs and 5-LO-specific inhibitors.

MATERIALS AND METHODS

A total of 45 nonconsecutive surgical cases of colorectal adenocarcinomas obtained from the same number of patients (19 females [42%] and 26 males [58%]; age range, 36-89 years; mean age, 68.2 years) were selected from the files of the Department of Human Pathology of the University of Messina, Italy. Selection was based on clinical records, so that patients who had received preoperative neoadjuvant therapy were excluded from the study.

For each case, the age and sex of the patient, the localization and size of the tumor, as well as the clinical and pathologic stage, were considered. Histologic typing, grading, and tumor staging were based on the criteria of the World Health Organization classification and TNM system. According to tumor localization, samples were classified as "right sided" (localized in the cecum or in the ascendant or transverse colon) and "left sided" (set in the descending or in the sigmoid colon or in the rectum). On the basis of tumor size, 2 groups were identified, the first one comprising tumors 4 cm or smaller and the second one consisting of tumors larger than 4 cm. Local invasion was also classified into 2 groups, pT1-T2 and pT3-T4. Furthermore, cases were subdivided into 2 groups on the basis of their histologic grades, the first group comprising grade 1 and grade 2 cases, and the second group consisting of grade 3 adenocarcinomas. Finally, the patients were subdivided into 2 categories on the basis of their age at the time of surgical operation, with the age median value (70 years) used as the cutoff value. Data on tumor recurrences were available in 13 (29%) of 45 cases.

In the adenocarcinomas of the cohort, the tumor neoangiogenesis was quantified through the assessment of microvessel density (MVD) performed using the immunohistochemical detection of the specific marker for neoangiogenesis, CD105 (endoglin).

Immunohistochemistry

Immunohistochemical procedures were performed on formalin-fixed, paraffin-embedded, sequential tissue sections obtained from each representative paraffin block.

Briefly, sections were deparaffinized in xylene and rehydrated using graded alcohol and deionized water. Endogenous peroxidase activity was preliminarily blocked with 3% [H.sub.2][O.sub.2] for 15 minutes, and then normal sheep serum was applied for 30 minutes to prevent nonspecific adherence of serum proteins. For CD105 epitope retrieval, the specimens were pretreated with proteinase K (S3020, Dako Cytomation, Glostrup, Denmark) at room temperature for 15 minutes, whereas 5-LO antigen was unmasked by microwave oven pretreatment in 10mM sodium citrate buffer, pH 6.0, for 3 cycles X 5 minutes. Sections were successively incubated at 4[degrees]C overnight with the primary monoclonal antibodies against CD105 (working dilution 1:50; clone SN6h, Dako Corporation) and 5-LO (working dilution 1:100; Cayman Chemical Company, Ann Arbor, Mich); the bound primary antibody was visualized by avidin-biotin-peroxidase detection using the Vectastain Rabbit/Mouse Elite Kit (Vector Laboratories, Peterborough, England) according to the manufacturer's instructions. To reveal the immunostaining, the sections were incubated in darkness (18) for 10 minutes with 3-3'diaminobenzidine tetrahydrochloride (Sigma Chemical Company, St Louis, Mo) in the amount of 100 mg in 200 mL 0.03% hydrogen peroxide in phosphatebuffered saline solution. Nuclear counterstaining was performed by Mayer hemalum. Specificity of the binding was assessed by several controls: (1) omitting the primary antiserum, (2) replacing it with normal rabbit serum or, in the case of 5-LO immunoreaction, (3) previously adsorbing it with the homologous antigen (360402, Cayman Chemical).

Alveolar macrophages of human lung surgical samples were used as the positive controls for the 5-LO immunoreaction. (19) Tissue sections of renal cell carcinoma known to express CD105, as well as the syncytiotrophoblast within specimens of human term placenta, were used as the positive controls for the CD105 immunoassay. (20, 21)

Quantification and Statistics

The quantification of microvessels was performed according to the procedure described by Weidner et al. (22) The assessment was done within the cancerous mass, excluding all the necrotic and ulcerated areas and the lymphomonocyte infiltration. The 3 most vascularized areas detected by CD105 were initially selected (so called hot spots) under a X40 field. Microvessels were then counted in each of these areas under a X400 field. Only those vessels with a clearly defined lumen were counted. The mean value of three X400 field (0.30 [mm.sup.2]) counts was recorded as the MVD of the section. The MVD value was then converted into the mean number of microvessels per [mm.sup.2] for the statistical analyses. The vessels were counted using a Zeiss Axioplane microscope by 2 independent observers blinded to the clinicopathologic data.

The 5-LO immunostaining pattern was independently evaluated on parallel sections by light microscopy using a X 20 and a X40 objective lens and a X10 eyepiece. Assessment was performed by 2 pathologists who were blinded to the other immunohistochemical results. The 5-LO immunoexpression was semiquantitatively measured. As we previously described,14 staining intensity was graded as weak (1), moderate (2), or strong (3), whereas the stained area, recorded as the percentage of positive cells in the tissue section, was assessed by providing the following values: 0 (<10%), 1 (10%-40%), 2 (41%-70%), or 3 (>70%). In instances of interobserver disagreement, a consensus was reached using a double-headed microscope. Then, a 5-LO intensity distribution score was generated for each case by summing the values of the stained area and staining intensity. Cases displaying an intensity distribution score less than 1 were considered to be negative for 5-LO immunoexpression. The median intensity distribution score value (5-LO intensity distribution score median value, 3) was used as the cutoff to subdivide cases with a low (5-LO intensity distribution score, 1-3) and a high (5-LO intensity distribution score, 4-6) 5-LO expression.

The Mann-Whitney test was used to explore whether a statistically significant difference in the MVD counts existed between cases with a low and a high 5-LO expression amount. Then, the correlation between the MVD counts and 5-LO intensity distribution scores was further investigated by performing the Pearson correlation test. Finally, the correlations between the clinicopathologic variables and the MVD or 5-LO intensity distribution scores were analyzed through Mann-Whitney and [chi square] tests, respectively.

A P value <.05 was considered to be statistically significant. Data were analyzed using the SPSS package version 15.0 (SPSS Inc, Chicago, Ill).

RESULTS

Clinicopathologic characteristics and immunohistochemical data of the 45 analyzed colorectal adenocarcinomas are shown in Table 1.

CD105 immunoreactivity was found in 100% of cases, with a variable number of intratumoral labeled vessels (Figure 1). In 64% (29/45) of cases, a positive immunoreaction was also evidenced in the vessels within the normal colonic mucosa adjacent to the tumor (Figure 2), but not in those within the mucosa at distance from the tumor.

A variable positive immunoreaction for 5-LO was evident in the neoplastic cells of 45 (100%) of 45 analyzed adenocarcinomas (Figure 3), whereas the negative controls as well as the normal colonic mucosa did not express 5-LO. Specifically, a low 5-LO immunohistochemical expression (5-LO intensity distribution score, 1-3) was encountered in 27 (60%) of 45 colorectal adenocarcinomas, whereas 18 (40%) of 45 cases were characterized by a high 5-LO (5-LO intensity distribution score, 4-6) immunoexpression.

Significantly higher MVD counts were evident in the adenocarcinomas that displayed a high 5-LO intensity distribution score in comparison with those showing a low 5-LO intensity distribution score (P = .02; Table 2). The Pearson correlation test also revealed a positive significant correlation between the MVD values and the 5-LO intensity distribution scores (r = 0.2986, P = .04;Figure 4).

On the contrary, no statistically significant correlations were found between MVD and the various clinicopathologic characteristics of the tumors (Table 2), whereas a significant correlation was recorded between high 5-LO intensity distribution scores and the presence of lymph node metastases (P = .02) or an advanced stage (stages III-IV) of the disease (P = .04; Table 3).

COMMENT

Neoangiogenesis, the formation of new blood vessels, plays an important role in the growth and progression of tumors. (23, 24) Indeed, as long as the neoplastic mass is supplied only by the host vessels, it retains a limited volume and a low doubling time. By contrast, when the formation of new capillaries is induced by the neoplastic cells through the secretion of proangiogenic factors, the nutrients provided by the newly formed vessels allow a more rapid growth and the progression of the tumor. (25, 26)

Tumor angiogenesis is considered to be dependent on the balance between angiogenic and antiangiogenic regulators. (24)

The LO enzymes, which are upregulated in a number of malignancies, (8, 10, 13-16, 27) have been evidenced as key promotors of tumor neoangiogenesis via modulation of the vascular endothelial growth factor expression. (3, 4, 27, 28)

Indeed, with reference to 5-LO, in vitro studies (3) have shown that the transfection of a 5-LO construct in mesothelioma cell lines results in the increase of their vascular endothelial growth factor release, whereas a significant decrease in vascular endothelial growth factor levels is obtained by either the transfection of a 5-LO antisense oligonucleotide and the use of 5-LO inhibitors in these tumor cells. (3) In addition, Ye and colleagues (17) have suggested that 5-LO behaves as an inductor of angiogenesis in colorectal cancer; indeed, they have demonstrated the significant reduction in angiogenesis as a consequence of the specific inhibition of 5-LO activity in cigarette smoke-induced colorectal cancer. (17)

[FIGURE 1 OMITTED]

[FIGURE 2 OMITTED]

In the present study, we sought to analyze 5-LO's putative role in colorectal cancer neoangiogenesis by evaluating the correlations between 5-LO immunoexpression amount and the number of intratumoral newly formed vessels recognized by CD105 staining in a series of human sporadic colorectal adenocarcinomas. CD105, a 180-kd homodimeric transmembrane glycoprotein, (29) was chosen as the endothelial marker for the MVD assessment. It is predominantly expressed on the endothelial cells of newly formed vessels in tissues undergoing active angiogenesis, such as tumors (30, 31); moreover, CD105 was already demonstrated to be a more specific and sensitive marker for tumor neoangiogenesis than the commonly used panendothelial markers, such as CD31, in colorectal cancer. (32-34) In view of the fact that CD105 is also expressed by the vessels of regenerating or inflamed tissues, (35) we did not count the vessels in the necrotic and ulcerated areas of the tumor specimens when we quantified MVD. Even more, we only counted vessels with a clearly defined lumen, so as to avoid adding to the vessel count the intratumoral macrophages, which are known to express CD105. (36)

We documented CD105 immunoreactivity, with a variable number of labeled vessels, in 100% of the analyzed adenocarcinomas. The vessels present in the normal colonic mucosa in the invasive front of the tumor were also stained by the CD105 antibody in a percentage of cases, whereas no immunoreaction was present in those within the normal distant mucosa, thus confirming that CD105 is a highly specific marker for neoangiogenesis. (34, 37, 38)

As we previously demonstrated14 in accordance with other studies, (15-17) 5-LO was expressed in the neoplastic cells of the analyzed adenocarcinomas but not in the adjacent normal epithelium, which is in line with its putative role in colorectal carcinogenesis.

When 5-LO expression was correlated with the MVD counts, a strong relationship was found between the 5-LO amount and the number of newly formed vessels. When cases were stratified on the basis of their 5-LO intensity distribution scores, a significantly higher MVD value was found in 5-LO highly expressing colorectal adenocarcinomas in comparison with 5-LO lowly expressing tumors. Moreover, 5-LO intensity distribution scores appeared to be significantly positively correlated with the MVD counts. These results further suggest that 5-LO expression, which is acquired during carcinogenesis, may be one of the factors that regulate the formation of new blood vessels in colorectal adenocarcinoma.

[FIGURE 3 OMITTED]

[FIGURE 4 OMITTED]

In the present study, we also investigated the correlations between the MVD values and the clinicopathologic parameters, such as the patients' age and sex, or the size, the site, histologic grade, pTNM stage, and recurrences of the tumors. In contrast with the results achieved by other authors, who also used the CD105 antibody for the MVD assessment in colorectal adenocarcinoma, (33, 34, 39) we did not find any correlation between the MVD of the tumors and the mentioned parameters.

On the other hand, in line with the results that we had already achieved in a previous study, (14) a high 5-LO intensity distribution score was significantly associated with the development of lymph node metastases and with an advanced stage of the tumors of our cohort. Therefore, we may speculate that 5-LO stimulates not only the neoangiogenesis but also the lymphangiogenesis in colorectal cancer. Nonetheless, this topic remains to be more deeply investigated.

In conclusion, our study gives evidence of the existence of a relationship between 5-LO expression and tumor neoangiogenesis in colorectal cancer, thus suggesting that 5-LO may modulate the formation of new blood vessels in this tumor. Since CD105 is specifically expressed and limited to the intratumoral newly formed blood vessel, it would be interesting to evaluate in future studies whether therapies targeting CD105 that combine 5-LO-specific inhibitors and vaccines (40) may be useful in reducing the blood supply and, consequently, the growth and progression, of colon cancer.

We gratefully acknowledge C. M. Nunnari, MLT, for her precious technical assistance.

Accepted for publication April 3, 2008.

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Valeria Barresi, MD; Enrica Vitarelli, Bbs; Giovanni Tuccari, MD; Gaetano Barresi, MD

From the Department of Human Pathology, University of Messina, Messina, Italy.

The authors have no relevant financial interest in the products or companies described in this article.

Reprints: Valeria Barresi, MD, Department of Human Pathology, Policlinico Univeristario G. Martino, Pad D -Via Consolare Valeria, 98125 Messina, Italy (e-mail: valeriabarresi@hotmail.com).
Table 1. Clinicopathologic Characteristics, CD105 Microvessel Density
(MVD) Values, and 5-Lipoxygenase (5-LO) Intensity Distribution Scores
of the 45 Analyzed Colorectal Adenocarcinomas *

Case                    Tumor            Tumor      Histologic
No.    Age, y/Sex     Size, cm       Localization      Grade

 1        63/M           >4              Left            2
 2        75/F           >4              Right           3
 3        61/M           >4              Left            2
 4        87/M        [less than         Left            2
                    or equal to] 4
 5        66/F           >4              Right           2
 6        47/M           >4              Left            2
 7        63/F           >4              Left            2
 8        86/F        [less than         Left            1
                    or equal to] 4
 9        60/M        [less than         Right           2
                    or equal to] 4
10        64/M           >4              Right           2
11        77/M        [less than         Right           2
                    or equal to] 4
12        62/M           >4              Left            2
13        79/M           >4              Left            2
14        58/M           >4              Left            2
15        71/F        [less than         Left            2
                    or equal to] 4
16        71/M           >4              Right           2
17        65/M        [less than         Left            2
                    or equal to] 4
18        59/M           >4              Right           2
19        71/M           >4              Left            2
20        78/M        [less than         Left            3
                    or equal to] 4
21        88/F        [less than         Left            2
                    or equal to] 4
22        71/M        [less than         Right           3
                    or equal to] 4
23        50/F        [less than         Left            2
                    or equal to] 4
24        76/M        [less than         Left            2
                    or equal to] 4
25        37/F           >4              Left            2
26        55/M           >4              Right           2
27        63/F        [less than         Left            3
                    or equal to] 4
28        63/M           >4              Left            3
29        81/M        [less than         Right           2
                    or equal to] 4
30        84/F           >4              Right           2
31        36/M           >4              Left            2
32        54/F           >4              Left            2
33        68/M           >4              Right           2
34        62/M           >4              Right           3
35        73/M        [less than         Right           3
                    or equal to] 4
36        71/M           >4              Right           2
37        86/M           >4              Left            2
38        89/F           >4              Right           1
39        67/F           >4              Left            2
40        76/F           >4              Right           2
41        73/F           >4              Left            2
42        71/F        [less than         Right           2
                    or equal to] 4
43        68/F           >4              Left            2
44        76/F           >4              Left            3
45        70/F           >4              Right           3

                                                       5-LO Intensity
Case              Recurrences         CD105 MVD         Distribution
No.      pTNM        (DFI)      (vessels/[mm.sup.2])       Score

 1     T2 N0 M0       NA                67.6                  4
 2     T2 N0 M0       NA                18.6                  2
 3     T4 N2 M0       NA                47.6                  5
 4     T2 N0 M0       NA                60                    4
 5     T2 N0 M0       No                12                    1
 6     T3 N0 M0       No                23.3                  3
 7     T2 N0 M0       NA               140                    4
 8     T1 N0 M0       NA                20                    2
 9     T2 N0 M0       NA                12                    3
10     T3 N1 M0       No                72.2                  4
11     T2 N0 M0       NA                38.6                  3
12     T2 N0 M0       Yes               16.6                  4
13     T3 N1 M1       No                39                    2
14     T3 N1 M1       No                48.66                 2
15     T4 N1 M0       No                37.6                  4
16     T2 N0 M0       No                38.8                  3
17     T2 N1 M0       NA               105.5                  6
18     T2 N0 M0       NA                30                    3
19     T2 N0 M0       NA                12.22                 2
20     T3 N1 M1       NA                56.6                  1
21     T3 N0 M0       NA                 1.1                  1
22     T2 N0 M0       NA                76.6                  4
23     T4 N1 M1       Yes               16.6                  5
24     T2 N0 M0       NA                24.4                  2
25     T4 N0 M0       NA                10                    3
26     T2 N0 M0       NA                31                    1
27     T2 N0 M0       NA                32                    1
28     T3 N2 M0       No                 1.3                  6
29     T2 N0 M0       NA                76.6                  2
30     T2 N1 M0       NA                10                    1
31     T3 N1 M0       NA                47.77                 5
32     T4 N0 M0       No                10                    3
33     T4 N1 M0       Yes               13.3                  2
34     T2 N0 M0       NA               101                    4
35     T3 N2 M1       NA                23.3                  3
36     T2 N0 M0       NA                23.3                  3
37     T3 N0 M0       NA                 4.43                 5
38     T2 N0 M1       NA                40                    3
39     T3 N0 M0       NA                16.6                  3
40     T2 N1 M0       NA               136.6                  4
41     T4 N1 M0       No                23.3                  5
42     T2 N0 M0       NA                15.3                  1
43     T3 N1 M0       NA                11                    4
44     T2 N0 M0       NA                80                    2
45     T3 N2 M0       NA                85.3                  5

* DFI indicates disease-free internal; NA, not available.

Table 2. Statistical Correlations Between Microvessel
Density (MVD) and Clinicopathologic Parameters and
5-Lipoxygenase (5-LO) Intensity Distribution Score
Analyzed With Mann-Whitney and
Kruskal-Wallis Tests

Variable             n    Mean Rank    P

Sex                                   .15
  Male               26     25.38
  Female             19     19.74
Age y                                 .48
  [less than         23     21.65
  or equal to] 70
  >70                22     24.40
Size, cm                              .70
  [less than         15     24.07
  or equal to] 4
  >4                 30     22.47
Localization                          .40
  Left               26     21.62
  Right              19     24.89
Grade                                 .15
  1-2                36     21.58
  3                   9     28.67
T                                     .08
  T1-T2              25     26.06
  T3-T4              20     19.18
N                                     .38
  N0                 28     21.67
  N1-N2              17     25.17
M                                     .47
  M0                 39     22.44
  M1                  6     26.54
pTNM stage                            .30
  I-II               27     21.37
  III-IV             18     25.44
5-LO intensity                        .02
distribution score
  Low (1-3)          27     19.44
  High (4-6)         18     28.33
Recurrences                           .28
  No                 10      7.60
  Yes                 3      5.00

Table 3. Statistical Correlations Between
5-Lipoxygenase (5-LO) Intensity Distribution Scores
and Clinicopathologic Parameters Examined By [chi square] Test

                       5-LO Intensity
                     Distribution Score

Variable            Low (1-3)   High (4-6)   [chi square]    P

Sex
  Male                  12          7
  Female                15         11            0.004      .95
Age y
  [less than            11         12
  or equal to] 70
  >70                   16          6            1.960      .16
Size, cm
  [less than            10          5
  or equal to] 4
  >4                    17         13            0.104      .74
Localization
  Left                  13         13
  Right                 14          5            1.674      .20
Grade
  1-2                   22         14
  3                      5          4            0.006      .93
T
  T1-T2                 17          8
  T3-T4                 10         10            0.844      .35
N
  N0                    21          7
  N1-N2                  6         11            5.393      .02
M
  M0                    22         17
  M1                     5          1            0.019      .89
pTNM stage
  I-II                  20          7
  III-IV                 7         11            4.201      .04
Recurrences
  No                     6          4
  Yes                    1          2            0.215      .55
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