BioMed Research International

BioMed Research International / 2015 / Article
Special Issue

Molecular Markers in the Diagnosis and Treatment of Cancer

View this Special Issue

Research Article | Open Access

Volume 2015 |Article ID 131685 | 13 pages | https://doi.org/10.1155/2015/131685

Ki-67 Expression in CRC Lymph Node Metastasis Does Not Predict Survival

Academic Editor: Konstantinos Arnaoutakis
Received04 Dec 2014
Accepted02 Feb 2015
Published13 Sep 2015

Abstract

Colorectal cancer is one of the most common malignancies and a leading cause of cancer death worldwide. Molecular markers may improve clinicopathologic staging and provide a basis to guide novel therapeutic strategies which target specific tumour-associated molecules according to individual tumour biology; however, so far, no ideal molecular marker has been found to predict disease progression. We tested Ki-67 proliferation marker in primary and lymph node metastasis of CRC. We observed a statistical significant difference between the positive rates of neoplastic cells positively stained by Ki-67 in both sites, with remarkable increased number of Ki-67 positive cells in primary tumor cells compared to cancer cells that invaded lymph nodes. We can speculate that the metastatic CRC in lymph node can be more resistant to the drugs that target cellular division.

1. Introduction

Colorectal cancer (CRC) is one of the most common malignancies and a leading cause of cancer death worldwide [15]. European countries rank the highest in the global statistics, in terms of both CRC incidence and mortality [4, 6], although in recent years, a decline in CRC mortality rates has been observed, mostly due to improvement in earlier diagnosis and treatment [4, 7].

In Portugal, official data revealed that CRC is the second most common type of cancer, in both men and women, and in 2008 it was responsible for 18.7% and 15.1%, respectively, of all cancer in Portugal [8]. Regarding mortality, unlike European data [9], there was an average increase of 3% from 2000 to 2005 [10] and in 2012 incidence and mortality rates are higher than European rates [11].

For CRC, the pathologic clinical stage is currently the single most important prognostic factor [14, 12, 13], correlating with long-term survival [4, 1417], although it does not fully predict individual clinical outcome [4, 1719]. This is particularly true for those tumours with intermediate stage disease (T3-T4N0M0) [19], where one-third of patients with tumour-free lymph nodes have recurrences, and therefore adjuvant chemotherapy may be beneficial [20]. In this group, carcinoma cells are not detected in lymph nodes by conventional staging methods in 24% of patients. So, lymphatic staging is essential to improve treatment of these patients, indeed one-third of the patients submitted to curative intent surgery die of local and/or distant tumour recurrence [4, 15]. Abdominal lymph nodes (38%) are the second most frequent site of metastasis (38%), just after liver, that is, the organ most frequently involved (38–60% of cases) and followed by lung (38%) and peritoneum (28%) [4, 13].

A common feature of all cancers is the imbalance that exists between the proliferative activity and cell death; therefore, the evaluation of cell proliferation rate may be interesting in the study and characterization of tumours [21]. Some molecules, such as the Ki-67 protein, permit this assessment and are used as markers of proliferation because Ki67 expression is dependent of cell division rate; thus, overexpression of these markers may suggest a disruption in the proliferation mechanism leading to the appearance of tumours [21].

Ki-67 protein, when used to evaluate the percentage of dividing cells, allows us to determine neoplastic growth [21] and has been documented to correlate with neoplastic progression [22] showing different levels of expression between normal mucosa, adenoma, and adenocarcinoma [23], verifying a progressive increasing of positive Ki-67 expression from the first (normal mucosa) to the last part of the tissue (adenocarcinoma) [22, 24, 25].

Other studies correlate Ki-67 with the degree of malignancy, tumour invasiveness [25, 26], metastatic potential [21], patient survival, and the risk of relapse [27, 28]. Thus, a high Ki-67 expression in tumour cells is assumed to correlate with a poor tumour differentiation [24, 26] and an increased infiltration of the bowel wall (pT) [26]. Micev et al. [29] demonstrated that there is an association between Ki-67 expression and a less effective response in patients undergoing chemotherapy.

Other correlations with clinical and pathological data were also investigated and a correlation was detected between a high expression of this protein and the following variables: patient’s age [25], tumour size [30], tumour localization [28], dysplasia degree [30], the presence of lymph node metastasis [22, 25, 28], and TNM [25] and Dukes [28] classification. Thus, the younger is the patient, the greater is the cellular proliferation and the lower is the degree of differentiation; with increasing malignancy a increased frequency of invasion and metastasis are observed and thus poorer prognosis [25].

In CRC, the analysis of colon adenomas has shown a different pattern for Ki-67 expression between normal tissue, adenomas, and adenocarcinomas, being limited to the crypts in normal tissue and expressed both in the crypts and in the surface epithelium in adenomatous polyps (tubular, villous) [31] and distributed homogeneously in adenocarcinoma [32]. Nussrat et al. [30] also observed an increase in Ki-67 rates being associated with the growth and rise of dysplasia in adenomas.

Studies on CRC indicated Ki-67 as a prognostic marker as the survival rate for patients with high expression of Ki-67 is significantly lower compared to those with low expression [25, 3335] and a predictor of CRC recurrence [36]. Also significant associations were found between higher index of Ki-67 and increased tumour penetration [35, 37], the presence of lymph node [22, 35] and distant [35] metastasis, advanced TNM stage [32, 35], highest degree of differentiation, and subtypes of adenocarcinoma other than mucinous [38].

However, not all studies are in agreement, and no correlations were observed with patient age, gender, tumour location [22, 30, 33, 39], and the type of adenoma [30] for some of them. Allegra et al. [40] described inverse associations, with a lower rate of Ki-67 to be associated with greater recurrence and worse overall survival and Jansson and Sun [39] did not find any associations between index Ki-67 and clinicopathological data or prognosis.

Regarding the use of Ki-67 in CRC lymph node metastasis, no information is available, and the only similar study found compares Ki-67 index in primary tumour with peritoneal metastasis and had observed a lower proliferative index in metastasis compared with the primary tumour [41]. However, in other types of cancer, in particular breast cancer, a higher Ki-67 index was found in lymph node metastasis than in primary tumours [4244], suggesting greater aggressiveness of these [42] and that the use of Ki-67 in lymph node metastasis may be important in selecting the appropriate treatment for certain subgroups of patients [45].

Therefore, given the limited information concerning Ki-67 index in CRC lymph nodes metastasis and primary tumour, this study becomes relevant to determine Ki-67 index in the primary tumour and, respectively, lymph nodes metastasis whilst trying to establish correlations with this and clinicopathological data and the patient’s prognosis.

2. Materials and Methods

2.1. CRC Tumour Series

Tissue samples and data from 672 patients treated in Hospital de Braga, Portugal, between January 1, 2005, and January 1, 2010, with CRC diagnosis were collected prospectively. Tumour localization was recorded and classified as colon and rectum (between anal verge and 15 cm at rigid rectoscopy).

The histological type of CRC was classified by two experienced pathologists and tumour staging was graded according to the TNM classification, sixth edition [46]. Tissue microarrays (TMAs) were constructed with the CRC series of formalin-fixed, paraffin-embedded tissues and analyzed by immunohistochemistry. Prior to tumour construction, hematoxylin and eosin sections were reviewed to select representative areas of the tumour and normal-adjacent tissue. Each case was represented in the TMA by at least two cores of 0.6 mm.

2.2. Lymph Node Metastasis Series

From the same series of colorectal cancer, patients with the diagnosis of CRC lymph node metastasis were selected and a series of 210 patients were also collected.

Additionally, 35 patients, with the diagnosis of CRC but without lymph node metastasis, were also selected for control of protein lymph node expression (stages T1 and T2/N0).

TMAs were constructed with the lymph node metastasis series of formalin-fixed, paraffin-embedded tissues and analyzed by immunohistochemistry. Each case was represented in the TMA by at least two cores of 0.6 mm.

The study protocol was approved by the Ethics Committee of Hospital de Braga and ICVS.

2.3. Immunohistochemistry

CRC and lymph nodes TMAs protein expression was evaluated by immunohistochemistry. Detailed information is given in Table 1. After the immunohistochemical procedure, the slides were evaluated and then photographed under a microscope.


Protein markerAntigen retrieval Peroxidase
inactivation
Detection systemAntibody
CompanyDilutionIncubation period

Ki-67Citrate Buffer 0.01 M pH = 6.03% H2O2 in methanol, 10 min.RTU Vectastain
ABC elite Reagent
GenNova1 : 200Overnight

For positive control of the expression of Ki-67 a sample of the skin was used (Figure 1).

2.4. Immunohistochemical Evaluation

The percentage of immunoreactive cells was determined (which was named the Ki-67 index), counting a total of 100 cells per section at ×20 magnification, and each one was assigned a score from 0 to 3, as previously described by Pinheiro et al. [47].

Immunoreaction final score was defined as the sum of both parameters and grouped as negative (0-1) and positive (≥2). Evaluation of protein expression was performed by blind analysis by two observers and discordant cases were discussed in a double-head microscope in order to determine a final score.

2.5. Statistical Analysis

All data were analyzed using the Statistical Package for the Social Sciences, version 19.0 (SPSS Inc., Chicago, IL, USA). All comparisons were examined for statistical significance using Pearson’s chi-square () test and Fisher’s exact test (when ), with the threshold for significance . Survival curves were determined for overall survival by the Kaplan-Meier method and log-rank test.

Expression differences between lymph node metastasis and primary CRC were tested with McNemar test, with the threshold for significance .

3. Results

3.1. Ki-67 Expressions in CRC Samples

A total of 672 samples were organized into TMAs, including tumour and normal adjacent epithelium (). Sections were evaluated for immunoexpression and the obtained results are given in Table 2, which summarizes the frequency of Ki-67 expression in tumour cells and .


Protein markerImmunoreaction
Ki-67Positive (%)

CCR
 NAE14034 (24.3)<0.001
 Tumour506345 (68.2)
Lymph node
 Normal22 (100.0) 0.502*
 Metastasis10960 (55.0)

NAE: normal adjacent epithelium;: total number of cases with and without expression of Ki-67; positive (%): total number of cases with expression of Ki-67 and respective percentage.
*Comparisons were examined for statistical significance using Fisher’s exact test (when < 5).

We observed that 68.2% () of the samples of tumour tissue were positive for Ki-67, as compared to 24.3% () of samples of the samples of . Thus, it was concluded that the Ki-67 expression is significantly higher in tumour tissue (), such as is shown in Table 2.

Figure 2 shows representative cases of positive staining for Ki-67 in tumour cells and in .

3.2. Associations between Ki-67 Expressions in CRC Tissues and Clinicopathological Data

The associations observed between the expression of Ki-67 in CRC and the clinicopathological data are described in Tables 3 and 4.


Ki-67 in CRCKi-67 in lymph node metastasis
Positive (%)Positive (%)

Gender
 Male307212 (69.1)0.6757141 (57.7)0.438
 Female180121 (67.2)3819 (50.0)
Age
 ≤452314 (60.9)0.49174 (57.1)1.000*
 >45464319 (68.8)10256 (54.9)
Presentation
 Asymptomatic8863 (71.6)0.474198 (42.1)0.212
 Symptomatic 399270 (67.7)9052 (57.8)
Localization
 Colon353240 (68.0)0.7649050 (55.5)0.841
 Rectum13493 (69.4)1910 (52.6)
Macroscopic cancer type
 Polypoid253171 (67.6)0.1784520 (44.4)0.373
 Ulcerative11176 (68.5)3120 (64.5)
 Infiltrative3822 (57.9)116 (54.5)
 Exophytic3932 (82.1)127 (58.3)
 Vilosous22 (100.0)10 (0)
CEA (ng/mL)
 ≤10337229 (68.0)0.7506734 (50.7)0.757
 >107351 (69.1)2212 (54.5)

Comparisons were examined for statistical significance using Fisher’s exact test (when < 5).

Ki-67 in CRCKi-67 in lymph node metastasis
Positive (%)Positive (%)

Tumor size
 ≤4.5 cm279190 (68.1)0.5196539 (60.0)0.211
 >4.5 cm179127 (70.9)4019 (47.5)
Histological type
 Adenocarcinoma409281 (68.7)0.6658846 (52.3)0.483
 Mucinous adenocarcinoma5032 (64.0)138 (61.5)
 Invasive adenocarcinoma2418 (75.0)64 (66.7)
 Signet ring and mucinous42 (50.0)22 (100.0)
Differentiation
 Well differentiated209135 (64.6)0.0493819 (50.0)0.670
 Moderately differentiated208146 (70.2)4626 (56.5)
 Poorly differentiated4740 (85.1)2314 (60.9)
 Undifferentiated43 (75.0)11 (100.0)
Tumour penetration
 pT13423 (79.3)0.01322 (100.0)0.553
 pT25739 (68.4) 42 (50.0)
 pT3370252 (68.1) 9653 (55.2)
 pT42619 (73.1) 73 (42.9)
Spread to lymph nodes
 Absent 275188 (68.4)0.94095 (55.6)1.000*
 Present 198136 (68.7)8950 (56.2)
Venous vessel invasion
 Absent264179 (67.8)0.5112913 (44.8)0.110
 Present201142 (70.4)7446 (62.2)
TNM
 Stage I7554 (72.0)0.425
 Stage II181121 (66.9)
 Stage III152108 (71.1)7843 (55.1)0.978
 Stage IV 7045 (64.3)3117 (54.8)

Comparisons were examined for statistical significance using Fisher’s exact test (when ).

Analyzing the results in these tables, we found an association between the expression of Ki-67 and “tumour penetration” () and “tumour differentiation” ().

For “tumour penetration,” we observed a decreasing expression of Ki-67 from the pT1 (79.3%) to pT3 (68.1%) tumours and then a rise in expression for adenocarcinoma with invasion of other organs or structures (pT4) (73.1%).

Regarding “tumour differentiation,” we observed an increasing expression of Ki-67 from the well-differentiated to the undifferentiated tumours, namely, well differentiated (64.6%), moderately differentiated (70.2%), and poorly differentiated (85.1%). Conversely, undifferentiated tumours showed lower expression of Ki-67 compared to the degree of differentiation mentioned above.

We did not find any statistically significant relationship between clinicopathological data and Ki-67 index in CRC for the remaining assessed data.

3.3. Overall Survival Curves according to Ki-67 Expressions in CRC Tissues

No statistically significant association was observed for Ki-67 expression in CRC tissues ( for CRC, and and for colon cancer and rectal cancer evaluated separately, resp.), as observed in Figure 4.

Relatively to CRC, survival of patients that are negative for Ki-67 is 65.6% with a medium of survival of months after diagnosis, while Ki-67 positive patients present a survival of 62.3% with a medium of survival of months after diagnosis, such as is shown in Table 5.


Ki-67TissueDeaths
(%)
Median for survival time
95% confidence interval
Log-rank test

Negative CRC15453 (65.6)65.00 [59.49–70.52]0.321
Positive332125 (62.3)62.09 [58.06–66.12]

Negative Lymph node4917 (65.3)63.48 [53.18–73.79]0.131
Positive6230 (51.6)50.07 [41.06–59.08]

3.4. Ki-67 Expressions in Lymph Node Metastasis Samples

A total of 210 samples were organized into TMAs. Additionally 35 patients, with the diagnosis of CRC but without lymph node metastasis, were also selected for control of protein lymph node expression (stages T1 and T2 N0). Sections were evaluated for immunoexpression and the obtained results are given in Table 2, which summarizes the frequency of Ki-67 expression in “normal” lymph nodes and lymph node metastasis.

We observed that 55.5% () of the samples of lymph node metastasis were positive for Ki-67, compared to 100% () of samples of the samples of “normal” lymph nodes. No significant correlation was observed (), such as is shown in Table 2 and Figure 3.

3.5. Associations between Ki-67 Expressions in Lymph Node Metastasis and Clinicopathological Data

The associations observed between the expression of Ki-67 in lymph node metastasis of CRC and the clinicopathological data are described in Tables 3 and 4. Analyzing these tables, we did not find any statistically significant relationship between clinicopathological data and Ki-67 index in lymph node metastasis.

3.6. Overall Survival Curves according to Ki-67 Expressions in Lymph Node Metastasis

Relatively to overall survival, patients with negative Ki-67 index present a survival of 65.3% with a medium of survival of months after diagnosis, while Ki-67 index positive patients present a survival of 51.6% with a medium of survival of months after diagnosis, such as is shown in Table 5.

No statistically significant association was observed for Ki-67 expression in CRC lymph node metastasis tissues ( for CRC, and for colon cancer and rectal cancer evaluated separately, resp.); however, a tendency for the relationship between a positive Ki-67 index and a lower overall survival was observed, such as is shown in Figure 5.

3.7. Comparing Ki-67 Index Expressions in Lymph Node Metastasis and Primary Tumour

Table 6 represents a comparison between Ki-67 index in the primary tumour and the respective lymph node metastasis. Analyzing this table, it appears that 12 of the cases with negative Ki-67 index in the primary tumour have positive index in lymph node metastasis and that 29 of those with positive index in the primary tumour have a negative index in lymph node metastasis, for a total of 41 discordant cases. This means that there is a significant difference () between the index of Ki-67 in primary tumour and the respective lymph node metastasis. A smaller number of cases of positive Ki-67 index were also observed in lymph node metastasis (; 57.5%) than in the primary tumour (; 73.6%) as is schematized in Figure 6.


Ki-67 index in CRCKi-67 index in lymph node
NegativePositiveTotal (%)

Negative1612 28 (26.4)
Positive294978 (73.6)

Total (%)45 (42.5)51 (57.5)106 (100)

Comparisons were examined for statistical significance using McNemar test.

4. Discussion

The mechanisms that culminate in CRC development, growth, and metastization are still not fully understood. However, common to all cancers are the loss of cellular differentiation and the imbalance between proliferation and cell death; these processes, involved in carcinogenesis, due to its significance, are increasingly being targeted for study.

Ki-67 protein has been widely used as a marker of tumour proliferation [21, 48, 49], and several studies compare Ki-67 index with clinicopathological data and follow-up in CCR [22, 32, 3540]. With regard to Ki-67 index in lymph node metastasis, as far as we know, this is the first study realized in CRC and, the more similar that we found in literature is the study of Yamauchi et al. [41], which compares Ki-67 index in primary CRC tumours with the respective nodules of peritoneal metastization.

In this study, we determine immunohistochemical expression of Ki-67 protein in CRC samples and respective lymph node metastasis and intended to evaluate possible associations between these expressions and several clinicopathological parameters and patient survival. Further comparison was performed between Ki-67 index in CRC and respective lymph nodes metastasis.

Regarding Ki-67 expression in CRC samples and we observed a significant expression of Ki-67 in the first over the second (). These results were expected, since due to its role as a marker of cellular proliferation, a higher expression was expected in tumour tissue than in normal epithelium. These results were also demonstrated by Lin et al. [25].

The same analysis was made for lymph nodes metastasis and “normal” lymph nodes, but no significant correlation was observed (). Possible explanations are the fact that “normal” lymph nodes are not truly normal, but of patients with CRC without lymph nodes metastasis (T1 and T2/N0) so this is a bias to be considered since they may already be under the influence of the tumour environment. Another fact that may influence this result is the small size sample of the “normal” lymph node, so further studies need to be realized with bigger samples and normal lymph nodes for control.

When analyzing the correlation of Ki-67 expression in CRC with pathological data, we found an association between the expression of Ki-67 and “tumour penetration” () and “tumour differentiation” ().

For “tumour penetration,” we observed a decreasing expression of Ki-67 from the pT1 (79.3%) to pT3 (68.1%) tumours and then a rise in expression for adenocarcinoma with invasion of other organs or structures (pT4) (73.1%).

The decreased expression of Ki-67 with the increasing tumour penetration is conflicting since we would expect an increase in expression with increasing tumour wall penetration, as is observed for pT4. But as the Ki-67 is only a marker of cellular proliferation, other factors may influence this outcome.

Regarding “tumour differentiation,” we observed an increasing expression of Ki-67 from the well-differentiated to the undifferentiated tumours; conversely, undifferentiated tumours showed lower expression of Ki-67 compared to the degree of differentiation mentioned above.

As was observed by some authors [38, 50], the more undifferentiated is the tumour, the higher is the rate of cell proliferation and therefore Ki-67 index. This is not consistent with our findings; however, since the lower expression in undifferentiated tumours may be explained by the small size of this sample, further studies with larger series of undifferentiated tumours are necessary.

When analyzing the correlation of Ki-67 expression in lymph node metastasis with pathological data, any statistically significant relationship was observed. In the literature, no other studies realized in lymph node metastasis of CRC were found, and similar results were observed by Jansson and Sun [39] on the primary tumour but contradict the other studies analyzed [22, 25, 2830, 32, 3540].

In our series, we have not observed association between Ki-67 expression and patient’s survival, for CRC () and for lymph node metastasis (), series and the same was true when we considered separately colon cancer and rectal cancer. This was corroborated by the observations of Jansson and Sun [39]; however, these findings contradict, in part, the report of Valera et al. [35] that studied primary CRC tumours, and also contradict studies made with lymph node metastasis from breast cancer [4345] and prostate cancer [51], where a higher ki-67 index was associated with a worse patient survival.

Finally, we found association () between Ki-67 index in primary tumour and the respective lymph node metastasis and also observed that Ki-67 index was more often positive in the primary tumour than in the respective lymph node metastasis. This result is consistent with the study carried out by Yamauchi et al. [41] to compare Ki-67 index in CRC primary tumour and respective nodules of peritoneal dissemination, which, as in the present study, present a greater proportion of proliferating cells in the primary tumour than in the nodules of peritoneal dissemination, not advancing; however, there is no explanation for this finding. Distinct results were observed for similar studies realized in breast cancer [4244], where lymph node metastasis presents higher ki-67 index than primary tumour, but also no explanation was mentioned. Recently, Jo and colleagues have found a significant difference of higher Ki-67 proliferation in nodal metastasis and primary gastric cancer [52]. Most of the criticism of Ki-67 evaluation is related to the differences of antibodies, slide background, retrieval protocols applied in preparing the immunoreaction, and the scores used to evaluate the significance of Ki-67 proliferation rates. This concern is pertinent and most of the works that evaluate this premise did not reach a consensus. Interesting, automated evaluation of the Ki-67 labelled preparation has been adjudicated as superior than manual analyses. Moreover, subdividing the cases in low and high proliferative rate improve the kappa correlation. Besides these advisements, the lack of standard protocols among the laboratories limits the clinical relevance of the works [53].

This difference between Ki-67 index in primary CRC tumour and respective lymph node metastasis may explain the absence of correlation with clinicopathological data and survival observed in this study as it shows that the proliferative profile of the primary CRC tumour is different from that of its metastasis. One possible explanation for the low proliferative index of lymph node metastasis as compared to primary tumour is that lymph node cannot represent an optimal proliferative environment, since it is known that nutrient and oxygen deprivation induces cell cycle arrest, leading to a decreased proliferation rate [54]. Another possible explanation may be due to of Ki-67 own usage as a marker of cell proliferation, since Ki-67 expression seems to be influenced by nutrient intake of the cell [48].

The smaller number of patients with a positive Ki-67 index in lymph node metastasis can also contribute to the poor prognosis attributed to the presence of lymph node metastasis [32, 50, 55, 56] in CCR, since most antineoplastic agents target proliferating cells, cells with a low proliferation rate are more resistant to such treatment [53], and this hypothesis was also stated by Cabibi et al. [44], relatively to the subgroup of patients with breast cancer and lower ki-67 index in lymph node metastasis than in primary tumour.

5. Conclusions

In this study, we evaluated the immunohistochemical expression of Ki-67 in CRC and respective lymph node metastasis and simultaneously try to determine its correlation with clinicopathological data and patient survival. From the results obtained, it was found that this protein has higher expression in tumour tissue, supporting the hypothesis of involvement of Ki-67 in CRC and its role as a proliferative marker. Furthermore, in CRC samples, the association between the expression of this protein and the degree of tumour differentiation and penetration was found which enables Ki-67 to be used as a potential prognostic factor in CRC.

Although we have not obtained statistical significant results for lymph node metastasis series we observed a statistically significant difference between the Ki-67 index of the primary tumour and the respective lymph node metastasis, which is more often positive in the primary tumour. These results show that lymph node metastasis is composed of proliferating cells slower than that present in the primary tumour and it is hypothesized that the lymph node might not constitute an optimal environment for tumour cells proliferation or that Ki-67 might not be the more suitable proliferative marker for use in lymph node metastasis. This result also raises the possibility that tumour cells in lymph nodes can be more resistant to chemotherapy treatments, thus contributing to the poor prognosis of these patients.

Conflict of Interests

The authors declare that they have no conflict of interests.

References

  1. S. Svagzdys, V. Lesauskaite, D. Pavalkis, I. Nedzelskiene, D. Pranys, and A. Tamelis, “Microvessel density as new prognostic marker after radiotherapy in rectal cancer,” BMC Cancer, vol. 9, article 95, 2009. View at: Publisher Site | Google Scholar
  2. G. Des Guetz, B. Uzzan, P. Nicolas et al., “Microvessel density and VEGF expression are prognostic factors in colorectal cancer. Meta-analysis of the literature,” British Journal of Cancer, vol. 94, no. 12, pp. 1823–1832, 2006. View at: Publisher Site | Google Scholar
  3. H. Brenner, M. Hoffmeister, and U. Haug, “Should colorectal cancer screening start at the same age in European countries? Contributions from descriptive epidemiology,” British Journal of Cancer, vol. 99, no. 3, pp. 532–535, 2008. View at: Publisher Site | Google Scholar
  4. S. F. Martins, R. M. Reis, A. M. Rodrigues, F. Baltazar, and A. Filho, “Role of endoglin and VEGF family expression in colorectal cancer prognosis and anti-angiogenic therapies,” World Journal of Clinical Oncology, vol. 2, no. 6, pp. 272–280, 2011. View at: Publisher Site | Google Scholar
  5. S. F. Martins, E. A. Garcia, M. A. M. Luz, F. Pardal, M. Rodrigues, and A. L. Filho, “Clinicopathological correlation and prognostic significance of VEGF-A, VEGF-C, VEGFR-2 and VEGFR-3 expression in colorectal cancer,” Cancer Genomics & Proteomics, vol. 10, no. 2, pp. 55–67, 2013. View at: Google Scholar
  6. M. Zavoral, S. Suchanek, F. Zavada et al., “Colorectal cancer screening in Europe,” World Journal of Gastroenterology, vol. 15, no. 47, pp. 5907–5915, 2009. View at: Publisher Site | Google Scholar
  7. C. Bosetti, F. Levi, V. Rosato et al., “Recent trends in colorectal cancer mortality in Europe,” International Journal of Cancer, vol. 129, no. 1, pp. 180–191, 2011. View at: Publisher Site | Google Scholar
  8. Roreno, Corpo Humano—Roreno—Registo Oncológico Regional do Norte, http://www.roreno.com.pt/p.
  9. M. Malvezzi, P. Bertuccio, F. Levi, C. La Vecchia, and E. Negri, “European cancer mortality predictions for the year 2013,” Annals of Oncology, vol. 24, no. 3, pp. 792–800, 2013. View at: Publisher Site | Google Scholar
  10. C. G. Pinto, A. T. Paquete, and I. Pissarra, “Colorectal cancer in Portugal,” European Journal of Health Economics, vol. 10, no. 1, pp. S65–S73, 2010. View at: Publisher Site | Google Scholar
  11. J. Ferlay, E. Steliarova-Foucher, J. Lortet-Tieulent et al., “Cancer incidence and mortality patterns in Europe: estimates for 40 countries in 2012,” European Journal of Cancer, vol. 49, no. 6, pp. 1374–1403, 2013. View at: Publisher Site | Google Scholar
  12. S. Gurzu, J. Jung, L. Azamfirei, T. Mezei, A. M. Cîmpean, and Z. Szentirmay, “The angiogenesis in colorectal carcinomas with and without lymph node metastases,” Romanian Journal of Morphology and Embryology, vol. 49, no. 2, pp. 149–152, 2008. View at: Google Scholar
  13. S. Cascinu, V. Georgoulias, D. Kerr, T. Maughan, R. Labianca, and M. Ychou, “Colorectal cancer in the adjuvant setting: perspectives on treatment and the role of prognostic factors,” Annals of Oncology, vol. 14, no. 2, pp. 25–29, 2003. View at: Google Scholar
  14. D. D. Alexander, J. Waterbor, T. Hughes, E. Funkhouser, W. Grizzle, and U. Manne, “African-American and Caucasian disparities in colorectal cancer mortality and survival by data source: an epidemiologic review,” Cancer Biomarkers, vol. 3, no. 6, pp. 301–313, 2007. View at: Google Scholar
  15. H. J. Calvo, G. D. Ortega, R. J. M. Pardo, M. A. J. López, and T. Cubo, “Biologia molecular del processo metastásico del cancer colorectal,” Cirugia Española, vol. 68, pp. 577–587, 2000. View at: Google Scholar
  16. S. Y. Zafar, A. P. Abernethy, D. H. Abbott et al., “Comorbidity, age, race and stage at diagnosis in colorectal cancer: a retrospective, parallel analysis of two health systems,” BMC Cancer, vol. 8, article 345, 2008. View at: Publisher Site | Google Scholar
  17. L. A. G. Ries, P. A. Wingo, D. S. Miller et al., “The annual report to the nation on the status of cancer, 1973–1997, with a special section on colorectal cancer,” Cancer, vol. 88, no. 10, pp. 2398–2424, 2000. View at: Publisher Site | Google Scholar
  18. R. S. Saad, Y. L. Liu, G. Nathan, J. Celebrezze, D. Medich, and J. F. Silverman, “Endoglin (CD105) and vascular endothelial growth factor as prognostic markers in colorectal cancer,” Modern Pathology, vol. 17, no. 2, pp. 197–203, 2004. View at: Publisher Site | Google Scholar
  19. C. Barozzi, M. Ravaioli, A. D'Errico et al., “Relevance of biologic markers in colorectal carcinoma: a comparative study of a broad panel,” Cancer, vol. 94, no. 3, pp. 647–657, 2002. View at: Publisher Site | Google Scholar
  20. A. J. Bilchik, M. DiNome, S. Saha et al., “Prospective multicenter trial of staging adequacy in colon cancer: preliminary results,” Archives of Surgery, vol. 141, no. 6, pp. 527–533, 2006. View at: Publisher Site | Google Scholar
  21. T. Scholzen and J. Gerdes, “The Ki-67 protein: from the known and the unknown,” Journal of Cellular Physiology, vol. 182, no. 3, pp. 311–322, 2000. View at: Publisher Site | Google Scholar
  22. K. Guzinska-Ustymowicz, E. Stepien, and A. Kemona, “MCM-2, Ki-67 and PCNA protein expressions in pT3G2 colorectal cancer indicated lymph node involvement,” Anticancer Research, vol. 28, no. 1, pp. 451–457, 2008. View at: Google Scholar
  23. D. Freitas, M. H. Goulão, E. Camacho et al., “Clinical relevance of proliferation biomarkers and p53 expression in rectal mucosa and sporadic colonic adenomas: a prospective study,” Hepato-Gastroenterology, vol. 49, no. 47, pp. 1269–1274, 2002. View at: Google Scholar
  24. H. A. Saleh, H. Jackson, G. Khatib, and M. Banerjee, “Correlation of bcl-2 oncoprotein immunohistochemical expression with proliferation index and histopathologic parameters in colorectal neoplasia,” Pathology & Oncology Research, vol. 5, no. 4, pp. 273–279, 1999. View at: Publisher Site | Google Scholar
  25. M. X. Lin, Z.-F. Wen, Z.-Y. Feng, and D. He, “Expression and significance of Bmi-1 and Ki67 in colorectal carcinoma tissues,” Ai Zheng, vol. 27, no. 12, pp. 1321–1326, 2008. View at: Google Scholar
  26. P. Dziegiel, J. Forgacz, E. Suder, P. Surowiak, J. Kornafel, and M. Zabel, “Prognostic significance of metallothionein expression in correlation with Ki-67 expression in adenocarcinomas of large intestine,” Histology and Histopathology, vol. 18, no. 2, pp. 401–407, 2003. View at: Google Scholar
  27. X. Z. Bai, J. R. W. Masters, N. O'Donoghue et al., “Prognostic markers in clinically localised prostate cancer,” International Journal of Oncology, vol. 14, no. 4, pp. 785–791, 1999. View at: Google Scholar
  28. W. X. Gao, J. G. Feng, Y. F. Yang et al., “PCNA in colorectal carcinoma and the prognosis,” Xian Dai Yu Fang Yi Xue, vol. 2, no. 34, pp. 225–229, 2007. View at: Google Scholar
  29. M. Micev, M. Micev-Cosić, V. Todorović et al., “Histopathology of residual rectal carcinoma following preoperative radiochemotherapy,” Acta Chirurgica Iugoslavica, vol. 51, no. 2, pp. 99–108, 2004. View at: Publisher Site | Google Scholar
  30. F. L. Nussrat, H. H. Ali, H. G. Hussein, and R. J. Al-Ukashi, “Immunohistochemical expression of ki-67 and p53 in colorectal adenomas: a clinicopathological study,” Oman Medical Journal, vol. 26, no. 4, pp. 229–234, 2011. View at: Publisher Site | Google Scholar
  31. S. Toru and B. Bilezikçi, “Early changes in carcinogenesis of colorectal adenomas,” West Indian Medical Journal, vol. 61, no. 1, pp. 10–16, 2012. View at: Publisher Site | Google Scholar
  32. Y. Hashimoto, M. Skacel, I. C. Lavery, A. L. Mukherjee, G. Casey, and J. C. Adams, “Prognostic significance of fascin expression in advanced colorectal cancer: an immunohistochemical study of colorectal adenomas and adenocarcinomas,” BMC Cancer, vol. 6, article 241, 2006. View at: Publisher Site | Google Scholar
  33. C. T. F. Oshima, K. Iriya, and N. M. Forones, “Ki-67 as a prognostic marker in colorectal cancer but not in gastric cancer,” Neoplasma, vol. 52, no. 5, pp. 420–424, 2005. View at: Google Scholar
  34. C. Evans, I. Morrison, A. G. Heriot et al., “The correlation between colorectal cancer rates of proliferation and apoptosis and systemic levels plus their influence upon survival,” British Journal of Cancer, vol. 94, no. 10, pp. 1412–1419, 2006. View at: Publisher Site | Google Scholar
  35. V. Valera, N. Yokoyama, B. Walter, H. Okamoto, T. Suda, and K. Hatakeyama, “Clinical significance of Ki-67 proliferation index in disease progression and prognosis of patients with resected colorectal carcinoma,” British Journal of Surgery, vol. 92, no. 8, pp. 1002–1007, 2005. View at: Publisher Site | Google Scholar
  36. H. L. De Menezes, M. J. Jucá, E. G. D. A. Gomes, B. L. B. B. P. Nunes, H. O. Costa, and D. Matos, “Analysis of the immunohistochemical expressions of p53, bcl-2 and Ki-67 in colorectal adenocarcinoma and their correlations with the prognostic factors,” Arquivos de Gastroenterologia, vol. 47, no. 2, pp. 141–147, 2010. View at: Publisher Site | Google Scholar
  37. C. Ghiţă, I. D. Vîlcea, M. Dumitrescu et al., “The prognostic value of the immunohistochemical aspects of tumor suppressor genes p53, bcl-2, PTEN and nuclear proliferative antigen Ki-67 in resected colorectal carcinoma,” Romanian Journal of Morphology and Embryology, vol. 53, no. 3, pp. 549–556, 2012. View at: Google Scholar
  38. U. Nabi, A. H. Nagi, and W. Sami, “Ki-67 proliferating index and histological grade, type and stage of colorectal carcinoma,” Journal of Ayub Medical College, Abbottabad, vol. 20, no. 4, pp. 44–48, 2008. View at: Google Scholar
  39. A. Jansson and X.-F. Sun, “Ki-67 expression in relation to clinicopathological variables and prognosis in colorectal adenocarcinomas,” APMIS, vol. 105, no. 9, pp. 730–734, 1997. View at: Publisher Site | Google Scholar
  40. C. J. Allegra, S. Paik, L. H. Colangelo et al., “Prognostic value of thymidylate synthase, Ki-67, and p53 in patients with Dukes' B and C colon cancer: a National Cancer Institute-National Surgical Adjuvant Breast and Bowel Project collaborative study,” Journal of Clinical Oncology, vol. 21, no. 2, pp. 241–250, 2003. View at: Publisher Site | Google Scholar
  41. H. Yamauchi, T. Suto, W. Kigure et al., “The progression potential of peritoneal dissemination nodules from gastrointestinal tumors,” International Surgery, vol. 96, no. 4, pp. 352–357, 2011. View at: Publisher Site | Google Scholar
  42. F. Buxant, V. Anaf, P. Simon, I. Fayt, and J. C. Noël, “Ki-67 immunostaining activity is higher in positive axillary lymph nodes than in the primary breast tumor,” Breast Cancer Research and Treatment, vol. 75, no. 1, pp. 1–3, 2002. View at: Publisher Site | Google Scholar
  43. D. Park, R. Kåresen, T. Noren, and T. Sauer, “Ki-67 expression in primary breast carcinomas and their axillary lymph node metastases: clinical implications,” Virchows Archiv, vol. 451, no. 1, pp. 11–18, 2007. View at: Publisher Site | Google Scholar
  44. D. Cabibi, V. Mustacchio, A. Martorana et al., “Lymph node metastases displaying lower Ki-67 immunostaining activity than the primary breast cancer,” Anticancer Research, vol. 26, no. 6B, pp. 4357–4360, 2006. View at: Google Scholar
  45. K. Tawfik, B. F. Kimler, M. K. Davis, F. Fan, and O. Tawfik, “Ki-67 expression in axillary lymph node metastases in breast cancer is prognostically significant,” Human Pathology, vol. 44, no. 1, pp. 39–46, 2013. View at: Publisher Site | Google Scholar
  46. F. L. Greene, D. L. Page, I. D. Fleming et al., Eds., AJCC Cancer Staging Manual, Springer, New York, NY, USA, 6th edition, 2002.
  47. C. Pinheiro, A. Longatto-Filho, C. Scapulatempo et al., “Increased expression of monocarboxylate transporters 1, 2, and 4 in colorectal carcinomas,” Virchows Archiv, vol. 452, no. 2, pp. 139–146, 2008. View at: Publisher Site | Google Scholar
  48. D. C. Brown and K. C. Gatter, “Monoclonal antibody Ki-67: its use in histopathology,” Histopathology, vol. 17, no. 6, pp. 489–503, 1990. View at: Publisher Site | Google Scholar
  49. J. Gerdes, U. Schwab, H. Lemke, and H. Stein, “Production of a mouse monoclonal antibody reactive with a human nuclear antigen associated with cell proliferation,” International Journal of Cancer, vol. 31, no. 1, pp. 13–20, 1983. View at: Publisher Site | Google Scholar
  50. Y. Kubota, R. E. Petras, K. A. Easley, T. W. Bauer, R. R. Tubbs, and V. W. Fazio, “Ki-67-determined growth fraction versus standard staging and grading parameters in colorectal carcinoma: a multivariate analysis,” Cancer, vol. 70, no. 11, pp. 2602–2609, 1992. View at: Publisher Site | Google Scholar
  51. M. Hosaka, Y. Takano, M. Iki, and et al, “Prognostic significance of Ki-67, p53, and Bcl-2 expression in prostate cancer patients with lymph node metastases: a retrospective immunohistochemical analysis,” Pathology International, vol. 48, no. 1, pp. 41–46, 1998. View at: Publisher Site | Google Scholar
  52. M. J. Jo, J. Y. Park, J. S. Song et al., “Biopathologic features and clinical significance of micrometatasis in the lymph node of early gastric cancer,” World Journal of Gastroenterology, vol. 21, no. 2, pp. 667–674, 2015. View at: Publisher Site | Google Scholar
  53. J. Harvey, C. Thomas, B. Wood et al., “Practical issues concerning the implementation of Ki-67 proliferative index measurement in breast cancer reporting,” Pathology, vol. 47, no. 1, pp. 13–20, 2015. View at: Publisher Site | Google Scholar
  54. O. Trédan, C. M. Galmarini, K. Patel, and I. F. Tannock, “Drug resistance and the solid tumor microenvironment,” Journal of the National Cancer Institute, vol. 99, no. 19, pp. 1441–1454, 2007. View at: Publisher Site | Google Scholar
  55. D. L. Longo, A. S. Fauci, D. L. Kasper, S. L. Hauser, J. L. Jameson, and J. Loscalzo, Eds., Harrison's Principles of Internal Medicine, McGraw-Hill, New York, NY, USA, 18th edition, 2012.
  56. T. E. Le Voyer, E. R. Sigurdson, A. L. Hanlon et al., “Colon cancer survival is associated with increasing number of lymph nodes analyzed: a secondary survey of intergroup trial INT-0089,” Journal of Clinical Oncology, vol. 21, no. 15, pp. 2912–2919, 2003. View at: Publisher Site | Google Scholar

Copyright © 2015 Sandra F. Martins et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

1430 Views | 428 Downloads | 3 Citations
 PDF  Download Citation  Citation
 Download other formatsMore
 Order printed copiesOrder

We are committed to sharing findings related to COVID-19 as quickly and safely as possible. Any author submitting a COVID-19 paper should notify us at help@hindawi.com to ensure their research is fast-tracked and made available on a preprint server as soon as possible. We will be providing unlimited waivers of publication charges for accepted articles related to COVID-19.