Correlation of Ki-67 Proliferating Index with Histological Stage and Grade in Colorectal Carcinoma

Correlation of Ki-67 Proliferating Index with Histological Stage and Grade  in Colorectal  Carcinoma

 *Sultana S,1 Islam N,2 Kabir E,3 Akhter S,4 Paul R,5 Shirin A,6  Khan AA,7 Jahan N8

Abstract
Colorectal carcinoma is the most  common cancer of gastrointestinal tract. It is the 3rd most  commonly  diagnosed  cancer  and  the  3rd  leading  cause  of  cancer  death. The  growth  of  tumor  in  colorectal   carcinoma  is  highly  variable  and  its  histological grading and  staging  has  important  role  in  diagnosis, treatment  and  overall  prognosis. To observe the Ki-67 expression in colorectal carcinoma and find out the possible correlation of Ki-67 proliferating index with histological grading and Duke’s staging. This  cross sectional  study  was  conducted  at  Sir Salimullah Medical College  from  July 2014  to June 2016. 98  patients  with colorectal carcinoma  enrolled  in  this  study  by  purposive  sampling. The H&E staining was done on paraffin embedded tissue sample. Ki-67 expression by IHC method. Ki-67 is a proliferation associated  nuclear  antigen  which  can be recognize by  MIB-1 monoclonal antibody, correlate with histological staging and grading in  colorectal carcinoma. Then tumours were graded according to WHO grading criteria and pathological staging was done according to Duke’s staging system and immunohistochemical staining for Ki-67 antigen expression. Results were subjected to statistical analysis. The results were considered to be significant when the P< 0.05. Ki-67 proliferative index was high in well and moderately differentiated adenocarcinoma but low in poorly differentiated which is statistically significant (p <0.05).  Ki-67 expression was high in early Duke’s stage A and B but low expression  in advanced Duke’s stage C (p>0.05). The result of  this  study will enlighten the clinician regarding  the need for  doing Ki-67 in  colorectal carcinoma which   would  contribute to better understanding  of the  treatment as well as prognosis.

[Journal of Histopathology and Cytopathology, 2018 Jul; 2 (2):125-133]

Key words: Colorectal carcinoma, Immunohistochemistry, Ki-67

  1. *Dr. Sahela Sultana, Assistant Professor, Department of Pathology, Dr. Sirajul Islam Medical College, Dhaka. sultana.sahela83@gmail.com
  2. Professor Dr. Nasimul Islam, Professor and Head, Department of Pathology, Sir Salimullah Medical College, Dhaka.
  3. Professor Dr. Enamul Kabir, Professor, Department of Pathology, Sir Salimullah Medical College, Dhaka.
  4. Salma Akhter, Assistant Professor, Department of Pathology, Universal Medical College, Dhaka.
  5. Rita Paul, Assistant Professor, Department of Pathology, Ibrahim Medical College, Dhaka.
  6. Afroz Shirin, Assistant Professor, Department of Pathology, Enam Medical College Savar, Dhaka.
  7. Abu Anis Khan, Assistant Professor, Department of Pathology, International Medical College, Dhaka.
  8. Nusrat Jahan, Lecturer, Department of Pathology, Sir Salimullah Medical College, Dhaka.

 

*For correspondence

Introduction

Colorectal carcinoma is the most common malignancy of gastrointesinal (GI) tract and is a major cause of morbidity and mortality worldwide.1 Colorectal cancer accounts for 10% of all cancers and it is the 2nd leading cause of death from malignancy in the industrialized world.2 There are nearly one million new cases of colorectal cancer diagnosed worldwide each year and half a million death.3 In 2013, there were an estimated 1,177,556 people living with colon and rectal cancer in the United States and the number of new cases of colon and rectal cancer was 41.0 per 100,000 men and women per year.4 Regarding age  incidence of colorectal carcinoma, recent reports show that in the USA it was the most frequent form of cancer among the person aged between 60-70 years and fewer than 20% of cases occurs before the age 50.5 The incidence of  colorectal cancer in Bangladesh is exactly not known but estimated population are approximately 15,10.1%.6 The distribution of colorectal carcinoma worldwide seems to be related to industrialization and socioeconomic standard and the incidence rate is higher in industrialized countries including Western Europe, Scandinavia and North America, whereas in the developing countries (sub-Saharan, Africa and Asia) the incidences appear to be lower.7

There are several staging system for colorectal carcinoma among these TNM and Duke’s staging systems are the most common way of staging and grading of colorectal carcinoma.8 The American Joint Committee (AJC) and the Union  for International  Cancer control (UICC) joined to produce the TNM system, which attempt to record clinical and pathological data, guide therapy and forecast prognosis, all in one.9 Whereas  the  classification   into  Duke’s stage A,B,C  cases   is  the  measurement  of  the  boundaries  reached and both   methods   permit the grouping  of  cases into favorable and unfavorable outcome.10 Histologically  the tumor is graded according to WHO grading criteria as well differentiated, moderately differentiated and poorly differentiated and the histological appearance of colorectal carcinoma may vary considerably with its major importance being related to prognosis.10

The use of monoclonal antibodies raised against specific antigens associated with the cell proliferation.11  Ki-67 is a proliferation associated nuclear antigen expressed in all cycling cell except resting cell in the G0 phase and it reflects cell in the S/G2+M phases in particularly.12  MIB-1 is a monoclonal antibody and it recognizes the Ki67 nuclear antigen in the formalin fixed paraffin embedded tissue section.13 Ki-67 expression is estimated as the percentage of tumors cells positively stained by the antibody with nuclear staining.12 The importance of Ki-67 as an indicator of tumor behavior and in colorectal cancer this index may be used as a marker of prognosis.12

The proliferative activity as measured by Ki-67 antibody is closely associated with histological  grade and stage.2 In 2008 Uzma Nabi, Nagi A H and Waqas Sami, Department of Pathology, University of Health Sciences, Lahore, Pakistan conducted a study on Ki-67 proliferating index and histological stage and grade  of colorectal carcinoma and observed that proliferative index is high in well and moderately differentiated  adenocarcinoma and in an early Duke’s stage (A or B).2 But Ki-67 proliferating activity is low in poorly differentiated tumour and  in an advanced Duke’s stage C.13,8 But there are some studies of Lanza, Cavazzinil  in 1990 and  Yokoyama N, Okomoto H in Japan in 2005 contradicting the above mentioned  association of Ki-67 versus grading and staging of colorectal carcinoma, they concluded that proliferating index of Ki-67 was increasing with increasing grade, stage.14, 15

Methods

This  cross sectional study was conducted among the 98 histopathologically diagnosed patients having colorectal carcinoma  over a period of two years in the department of surgery, Sir Salimullah Medical College. Study population were the patients having colorectal cancer underwent surgical  treatment in the department of surgery of Sir Salimullah Medical College. The proliferative activity as measured by Ki-67 antibody is closely associated with histological  grading  and staging of colorectal carcinoma. The representative sections were submitted for Immunohistochemical staining. The Ki-67 immunostaining were performed according to manufacture’s recommendation, using the MIB-1 clone (DAKO, Carpenteria, CA & Ventena Medical System, Tucson, AZ). Ki-67 immunostainined slides were examined via light microsccopy. Positive Ki-67 staining was observed brown granular nuclear staning. For Ki-67 scoring the most positive area of the tumor was selected avoiding foci of inflammation. The number of positive nuclei were counted in 500 tumor cells in a high power field. The average of the counts over the same slides was taken and expressed as the percentage of Ki-67 positive cells  in the tumor.

Statistical analysis were performed in SPSS statistical software program, Version 17.0. To correlate histological grading and staging of colorectal carcinoma with Ki-67 proliferating index were performed with  Mann- Whitney U test. The result were consider to be  significant when P<0.05. One way ANOVA followed by Bonferroni test was performed to compare between groups.

Results

98 cases were included in the present study.  Age incidence ranged from 28-78 years and their mean ± SD 47.38 ± 10.37. Maximum patients (30.6 %) were found in 41-50 years age group where M: F was 1.72: 1 (Table I). Regarding site of tumor more than 50% of patient had tumors in the left side of colon and in the rest of the cases tumors were present in caecum (26.5%; Table-II).  98 cases having different sizes of tumors and in most of the cases fifty nine cases (60.2%) tumors size were 3-4 cm and  their mean ± SD 4.8 ± 1.8 (Table-III). Different morphological types of tumor were observed in present study. In maximum forty cases (40.8%) the morphological types of tumors were ulcerative and only five cases (5.3%) tumors were infiltrative type. Rests were annular (33.5%) and polypoid (20.3%; Table IV).

In the present study, tumors were graded according to WHO grading system into well differentiated, moderately differentiated, poorly differentiated and grouped into A, B, C accordingly. The maximum cases 69 (70.4%) of colorectal carcinoma were well differentiated and their mean Ki-67 proliferating index was 47.83 ± 15.23. There were significant differences among the groups (A vs B vs C), when mean proliferating index of Ki-67 were compared among the three groups. The result was found statistically significant (P<0.05). However when compared in between groups only A vs C (between well differentiate & poorly differentiate) groups were found also statistically significant (P<0.05). In the present study, it was also observed that with increasing grade, Ki-67 proliferating index decreases (Table V) .

Regarding staging of the tumor (Duke’s staging) where  maximum  cases (44.8%) were in stage B1 and their mean Ki-67 proliferating index was 46.25±14.06. There was no significant differences among the stages and the result was not statistically significant (P>0.05). In the present study it was also observed that with increasing stage of the tumors, there was decreasing Ki-67 proliferating index (Table VI).

Table I: Distribution of patients according to age group with male female ratio (n=98)

Age groups         Frequency            M: F      Percentage
Total  Male Female
≤30

 

10    7    3           2.3:1       10.2
31-40 23   13   10           1.3: 1       23.5
 

41-50

 

30

 

19

 

11

 

1.72: 1

 

30.6

 

51-60

 

26

 

17

 

9

 

1.88: 1

 

26.5

 

≥60

 

9

 

9

 

0

 

9.00: 0

 

9.2

 

Total

 

98

 

Mean ± SD

 

 

47.01± 10.99

Range (Min-Max)                                              28 – 78

 

 

Table II: Distribution of tumors according to site (n=98)

Site of tumor Frequency (%)
Left side of colon
Sigmoid colon 34 (34.7)
Transverse colon 24 (24.6)
Rectum 14 (14.7)
Right side of colon
Caecum 26 (26.5)
Total 98 (100)

Table III: Distribution of patients according to tumor size (n=98)

Tumor size (cm) Frequency (%)
1 – 2 14 (14.3)
3 – 4 59 (60.2)
5 – 6 19 (19.4)
Mean ± SD 4.8 ± 1.8
Total 98 (100)

 

Table IV: Distribution of patients according to morphological types of tumor (n=98)

Morphology Frequency (%)
Ulcerative 40 (40.8)
Annular 33 (33.5)
Polypoid 20 (20.3)
Infiltrating 5 (5.3)
Total 98 (100)

 

Table V: Relation of Ki-67 proliferating index with histological grading (n=98)

Grading Frequency

n(%)

Ki-67 expression

(Mean ± SD)

P
Well differentiated (A) 69 (70.4) 47.83± 15.23
Moderate differentiate
(B)
15(15.3) 46.33 ± 18.07
Poor differentiated (C)  14(14.3) 35.35 ± 11.17
Statistical analysis
A vs B vs C 0.023*
A vs B 1.000ns
A vs C 0.019*
B vs C 0.162 ns

ANOVA followed by Bonferroni test was performed to compare between groups

Table VI: Relation of Ki-67 proliferating index with histological stage (n=98)

 

Duke’s staging

 

Frequency

n (%)

Ki-67 expression

(Mean ± SD)

p
Stage A 3 (3.3%) 53.33 ± 20.81 0.727ns
Stage B1 44 (44.8) 46.25 ± 14.06
Stage B2 20(20.5) 43.25 ± 14.06
Stage C1 14(14.8) 44.64 ± 21.07
Stage C2 17(17.5) 42.05 ± 13.69

 

ANOVA test was done to measure the level of significance.

 

 

 

 

 

Figure 1. Photomicrograph of histopathological section of well differentiated adenocarcinoma of colon (H&E method x100)

 

 

 

 

 

Figure 2. Photomicrograph of   well differentiated adenocarcinoma stained with Ki-67 immunostain showing high proliferative Index(x100).

 

 

 

 

Figure 3. Photomicrograph of histopathological section of poorly differentiated adenocarcinoma of colon ( H& E method x400).

 

 

 

 

 

Figure 4. Photomicrograph of   poorly differentiated adenocarcinoma stained with Ki-67 immunostain showing low Proliferative Index (x400).

Discussion

Colorectal cancer (CRC) is one of the most common malignancies and a leading cause of cancer death worldwide.16 The incidence of cancer colon and rectum was 41.0 per 100,000 men and women per year and the number of deaths was 15.1 per 100,000 men and women per year in Bangladesh.17 Management of  colorectal carcinoma depends on a number of  morphological and biological factors which include the pathological tumor stage (including involvement of lymph nodes, breach of serosa, distant spread etc.), primary tumor characteristics (including depth of tumor penetration in the bowel wall, histological subtype, histological grade and differentiation, venous and lymphatic invasion, perineural invasion and lymphocytic infiltration), status of surgical resection margins (free or involved).18 With assessment of tumor cell proliferation may predict tumor behavior.19 The aim of this study was to evaluate the proliferating index (PI) in formalin fixed, paraffin embedded tissue section of colorectal carcinoma, using monoclonal MIB-1 antibody (Ki-67) and to assess the relationship between proliferative index (PI) and various pathological findings in colorectal carcinoma including histological grade, and stage.

In the present study, the mean age of the patients was 47.01± 10.99 years and the highest number of malignant cases were seen in the 4th and 5th decades. Male female ratio was 1.72:1 and 1.88:1 in 4th and 5th decades accordingly. So, in present study, male were predominant than female. These findings were similar to other studies.20, 21

In 34.7% patients the tumors were located in sigmoid colon followed by caecum (26.5%), transverse colon (24.5%) and rectum (14.3%).  As per gross morphological type of cancer, maximum 40.8% were ulcerative and 33.5% were annular type and rest were polypoid (20.3%) and infiltrating type (5.3%). In one study, colon was more commonly affected site compared to rectum and most of the lesions were ulcerative.22 In another study rectum was the most common affected site and predominant lesions were annular.23

In present study, 60.2% of malignant cases had tumors size between 3-4 cm and 19.4% malignant cases had tumors size 5-6 cm. In a study in 2011 by Kornprat showed that maximum size of the tumor in colon cancer were in between 4.5 to 6.5 cm in majority of cases.24 In 2013 ASCO annual meeting showed a report where a study conducted on tumor size in colorectal cancer found that in majority of the cases the tumors sizes were in between 4-6 cm in colorectal carcinoma.25

In this present study it was observed that  the Ki-67 labelling index was high in grade I and grade II  compared to grade III. These results showed that proliferating index was low in poorly differentiated tumor compared to well and moderately differentiated tumor. When Ki-67 proliferating index was compared among three groups a statistically significant correlation was found in present study (p<0.05). These findings are similar to studies in Japan2 Pakistan12 and Finland.26 On the other hand, some of the studies showed contradictory result that Ki-67 proliferating index increased with increasing histological grade.26,27

In the present study, it was observed that Ki 67 labeling index was high in Duke’s stage A and B  and tumor in advanced stage (Duke’s C) have a low proliferating index  compared to tumors in  an early invasive stage (Duke’s A and B). Regarding the correlation of Ki-67 LI with staging, no statistically significant correlation was found. This result was similar with other studies in which they concluded that Ki-67 proliferating index was significantly lower in carcinoma in subserosa or deeper invasion compared to carcinoma with submucosa or muscularis mucosa invasion.2,8,26 In another study  contradictory results have been reported on associations of Ki-67 with prognosis and survival of colorectal tumors with a low proliferation index in Duke’s A and B tumors to be associated with survival impairment compared to those with high values.28 Tumors with high proliferative activity are known to be most responsive to radiotherapy and Willett and collaborators showed that radiation eradicates preferentially rapidly dividing cells in rectal cancer, whereas populations with slow proliferation show greater radioresistance.29

Conclusion

It is concluded that Ki-67 labeling index is high in well to moderately differentiated adenocarcinomas in an early Duke’s stage A or B compared to poorly differentiated adenocarcinomas, and  in an advanced Duke’s stage C. Thus Ki-67 proliferating index can be useful in a patient with colorectal carcinoma as a ancillary diagnostic support. Moreover, it may help in the prognostic evaluation of patient, survival as well as in considering them for post surgical treatment.

References

  1. Kumar V, Abul K Abbas, Nelson F, Jon C Aster. The Gastrointestinal Tract, Robbins and Cotran Pathologic Basis of Disease, 8th edition,Saunders, 2010;17: 822-826.
  2. Uzma N, Nagi A.H, Waqas S. Correlation of Ki 67 with histological grade, stage in colorectal carcinoma. Dept of pathology,University of health science, Lahore  Pakistan, J Ayub Med Coll, 2008;20(4):44-8.
  3. Bisgaard ML, Jager AC, Myrhoj T, Bernstein I, Nielsen FC. Hereditary non-polyposis colorectal cancer (HNPCC): phenotype-genotype correlation between patients with and without identified mutation. 2002; 20(1):20-7.
  4. Andrew RH, Hongmei N. Epidemiology of colorectal cancer. Int Journal of Molecular Epidemiology and  Genetics, 2016;7(3):105–114.
  5. Soreide K, Janssen MA, Soiland H, H, Baak APJ. Microsatellite  instability in colorectal cancer. Br J Surg, 2006; 93(4):395-406.
  6. Sayed A H, Richard S. Cancer control in Bangladesh. Jpn J Clin Oncol, 2013; 43(12):1159-1169.
  7. Alice G, Davies A, Liz G, Evropi T, Harry Cl. Estimating the incidence of colorectal cancer in  Sub-Saharan Africa: A systematic analysis. J Glob Health, 2012;2(2):020404.
  8. Ishida H, Sadahiro S, Suzuki T, Ishikawa K, Kamijo A, Tajima T. Proliferative, infiltrative, and metastatic activitie s in colorectal tumors assessed by MIB-1 anti-body. 2003;10(6):1741-5.
  9. Sobin LH, Godspodarowicz MK, Wittekind C. UICC TNM Classification of Malignant Tumours. 7th edition. Wiley-Blackwell, 2009;12(4):100-105.
  10. Gordon PH, Nivatvongs S. Missouri: Karen Berger, Principles and practice of surgery for the colon, rectum  and anus.1992;23(3):503–520.
  11. Mokrý J, Nĕmecek S. Immunohistochemical detection of proliferative cells’,Sb Ved Pr Lek Fak Karlovy Univerzity Hradci Kralove.1995;38(3):107-.130.
  12. Oshima FTC, Iriya K, Forones MN. Ki-67 as a prognostic marker in colorectal cancer but not in gastric cancer. Neoplasm, 2005;5(4):420-4.
  13. Parkin D, Bray F, Ferlay. Global cancer statistics. CA Cancer J Clinic, 2005;55(2):74-108.
  14. Lanza G Jr, Cavazzini L, Borghi L, Ferretti S, Buccoliero F and Rubbini M. Immunohistochemical assessment of growth fraction in colorectal adenocarcinomas with monoclonal antibody Ki-67. 1990;186 (1):608-18.
  15. Yokoyama N, Okamoto,Valentina V, Walter B,  Suda HT, Hatakeyama K. Clinical significance of Ki-67 proliferation index in disease progression and prognosis of patients with resected colorectal  carcinoma Version of   Record British Journal of Surgery Society  2005; 10:4858-15.
  16. World Health Organization Cancer Incidence in Five Continents. Lyon: The World Health Organization and The International Agency for Research on Cancer; WHO, Geneva, 2002. http://www.iarc.fr/en/publications/pdfs-online/epi/index.php.
  17. National Institutes of Health and Human service. What You Need To Know About Cancer of the Colon and Rectum. Bethesda, MD: U.S. Department of Health and Human Services & National Institutes of Health.2010. http://www.cancer.gov/cancertopics/types/colon-and-rectal.
  18. Ola Marzouk and John Schofield. Review of Histopathological and Molecular Prognostic Features in Colorectal Cancer Cancers (Basel). 2011;3(2):2767–2810.
  19. Valera V, Yokoyama N, Walter B, Okamoto H, Suda.T, Hatakeyama K. Clinical significance of Ki-67 proliferation index in disease progression and prognosis of patients with resected colorectal carcinoma’. Br J Surg 2005;92:1002-7.
  20. Bhagyalakshmi, Sreelekha A. Kasi Babu SV, Kumar P. Muralikrishnm Ki-67 proliferation index and clinicopathological patterns in colorectal carcinomas. Departments of   Pathology, Biochemistry, Surgery, Gastroenterology, Andhra Medical College, Visakhapatnam. 2015;4:119-28.
  21. El-Bolkainy TN, Sakr MA, Nouh AA, El-Din NH. A comparative study of rectal and colonic carcinoma: demographic, pathologic and TNM staging analysis. J Egypt Natl Canc Inst, 2006;18: 258-263.
  22. Missaouia N, Jaidaine L, Abdelkader AB, Beizig N, Anjorin A, Yaacoubi MT. . Clinicopathological patterns of colorectal cancer in Tunisia. Asian Pacific J Cancer Prev 2010; 11:1719-1722.
  23. Laishram RS, Kaiho N, Shimray R, Sorokhaibam BD, Pukhrambam P, Durlav CS. Histopathological evaluation of colorectal carcinomas status in Manipur, India. 2010; 8(1):5-8.
  24. Kornprat P, Pollheimer MJ, Lindtner RA, Schlemmer A, Rehak P, Langner C. Value of tumor size as a prognostic variable in colorectal cancer: a critical reappraisal. Am J Clin Oncol 2011; 34(1):43-9.
  25. ASCO 2013 Annual Meeting, Gastrointestinal Colorectal Cancer,ASCO university. https://meetinglibrary.asco.org/results/SessionTitle:/22Gastrointestinal/20/28Colorectal /Meeting:/222013/20ASCO.
  26. Eeva S, Salla P, Tero V, Peter J. Roberts, Karl-Owe Söderström. Increased proliferation activity measured by immunoreactive Ki-67 is associated with survival improvement in rectal/recto sigmoid cancer. Department of Oncology and Radiotherapy, Turku University Hospital, Finland 2005;11(21):3245–3249.
  27. Saleh HA, Jackson H, Khatib G, Banerjee M. Correlation of bcl2 oncoprotein immunohistochemical expression with proliferation index and histopathologic parameters in  colorectal neoplasia. Appl mmunohistochem Mol Morphol, 2002;8:175-182.
  28. Palmqvist R, Sellberg P, Oberg A, Tavelin B, Rutegård JN. Low tumour cell proliferation at the invasive margin is associated with a poor prognosis in Dukes’ stage B colorectal cancers. Br J Cancer 1999;79:577-581.
  29. Willett CG, Warland G, Hagan MP, Daly WJ, Coen J, Shellito PC, Compton CC. Tumor proliferation in rectal cancer following preoperative irradiation. J Clin Oncol 1999;13:1417-1424.

Leave a Reply

Your email address will not be published.