Fine Needle Aspiration Cytology in the Diagnosis of Focal Liver Lesions

Fine Needle Aspiration Cytology in the Diagnosis of Focal Liver Lesions

*Saem AM,1 Saha NK,2 Begum F,3 Hye AA,4 Islam N,5 Anam T6

 Abstract

Fine needle aspiration cytology (FNAC) assisted by cell block examination might be more accurate method for the definitive diagnosis of focal liver lesions (FLL). This study was designed to find out the role of FNAC in the diagnosis of FLLs in comparison to cell block preparations. This cross sectional observational study was carried out in the department of Pathology in collaboration with the department of Radiology & Imaging at Sylhet MAG Osmani Medical College. Study period was from 1 July, 2015 to 30 June, 2016. Clinically & radiologically diagnosed patients of focal liver lesions were study populations. The age of the study patients ranged from 15 to 80 years with a mean of 53.58 years. On FNAC, 10% cases were unsatisfactory, 8% cases were cystic lesion, 4% cases were benign tumor and 78% cases were malignant tumor. Among 39 malignant cases, 30.77% cases were hepatocellular carcinoma (HCC) and 69.23% cases were metastatic adenocarcinoma. Unsatisfactory samples were 18.18%, 6.82% were benign tumors and 75% were malignant tumors. Among the malignant lesions, 18.18% were HCC and 81.82% were metastatic adenocarcinoma. The sensitivity, specificity, positive predictive value (PPV), negative predictive value(NPV) and accuracy of FNAC in the evaluation of FLLs were 100%, 66.67%, 97.06%, 100% and 97.22%, respectively. The sensitivity, specificity, PPV, NPV and accuracy of FNAC in the detection of HCC were 66.67%, 85.18%, 50%, 92% and 81.82% respectively. FNAC of focal liver lesions has high sensitivity and accuracy in the detection of malignancy but it has low sensitivity in the detection of HCC. Cell block preparations were found superior to cytomorphology as immunostaining can be done on cell block preparations.

[Journal of Histopathology and Cytopathology, 2017 Jul; 1 (2):110-115]

 Key words: Focal liver lesions, FNAC, Cell block, Immunohistochemistry, HCC, and Metastatic carcinoma.

  1. *Dr. Abu Mohammad Saem, Lecturer, Department of Pathology, Comilla Medical College, Comilla. saemshampa@yahoo.com
  2. Naba Kumar Saha, Professor & Head, Department of Pathology, MAG Osmani Medical College, Sylhet.
  3. Ferdousy Begum, Associate Professor, Department of Pathology, Bangabandhu Sheikh Mujib Medical University, Dhaka.
  4. Azizul Qadar Md. Abdul Hye, Associate Professor Department of Pathology, MAG Osmani Medical College, Sylhet.
  5. Nazmul Islam, Assistant Professor, Department of Pathology, Army Medical College, Comilla.
  6. Tasmina Anam, Scientific Officer, Department of Pathology, Bangabandhu Sheikh Mujib Medical University, Dhaka.

 * For correspondence

 Introduction

A focal liver lesion (FLL) is a solid or cystic mass or area of tissue that is identified by radiological or imaging techniques as an abnormal part of the liver. It may be either a benign lesion such as focal nodular hyperplasia, hepatocellular adenoma and hepatic cyst or a malignant lesion such as hepatocellular carcinoma, cholangiocarcinoma, hepatoblastoma and metastatic carcinoma.1

Pathological examination is an important aspect in the evaluation of an FLL. FNAC is the preferred method for diagnosis of focal liver lesions and needle core biopsy (NCB) for evaluating diffuse liver diseases where architectural details are important.2 In recent years FNAC has emerged as an effective tool for diagnosis of a hepatic mass.

 

Cell blocks prepared from residual materials of fine needle aspirations can be useful adjuncts to smears for establishing a more definitive cytopathological diagnosis.3 Use of cell blocks improves diagnostic accuracy as it facilitates study of architecture details of multiple sections, use of special stains and immunohistochemistry.4

The distinction of moderately to poorly differentiated hepatocellular carcinoma from metastatic carcinoma may be a major problem for cytologists and this distinction is clinically important. Immunohistochemistry is required in this situation to differentiate hepatocellular carcinoma from metastatic carcinoma.5

With this background the study was designed to find out the role of FNAC in the diagnosis of focal liver lesions and to correlate its efficacy with cell block preparations using H&E and immunohistochemistry.

Methods

This cross sectional observational study was carried out in the department of Pathology in collaboration with the department of Radiology & Imaging at Sylhet MAG Osmani Medical College from 1 July, 2015 to 30 June, 2016. Clinically and radiologically diagnosed patients of focal liver lesions attending the department of Radiology & Imaging from different departments during the study period were the target population and those who fulfilled the inclusion and exclusion criteria were considered as study population. Patients of all ages and both sexes were included. Patients with bleeding diathesis, suspected liver abscess, hydatid cyst and hemangioma were excluded from the study. 22 gauge needle was placed in the lesion under ultrasound guidance and the material was aspirated with a 10 ml disposable syringe. After placing aspirates on the slides, thin smears were prepared by gentle friction of two slides. Then smears were fixed in 95% ethyl alcohol for at least 30 minutes and stained with Papanicolaou stain. After preparation of smears, the residual material was secured for clot preparation. It was then transferred into 10% formalin and processed as a cell block.6 Then, the cell blocks were cut at 5 micrometer thickness and were stained with Harri’s Haematoxylin and Eosin stain. From the paraffin block 3 micrometer sections were cut and stained for immunohistochemistry with Glypican-3 antibody. The immunohistochemistry was performed in the Immunohistochemistry Laboratory of Bangabandhu Sheikh Mujib Medical University (BSMMU) following their staining protocol. All the data were organized by using scientific calculator and Statistical Package for Social Science (SPSS) version 23.

 Results

The age of the study patients ranged from 15 to 80 years with a mean of 53.58 years (SD +15.32). Out of 50 cases, 33 (66%) were male and 17 (34%) were female with male to female ratio of 1.94:1. Among these patients, the highest number of patients 13(26%) were in the age group 51-60 years (Table I).

Table I: Age and sex distribution of study cases (n=50)

 

Age Groups (years) Male
No (%)
Female

No (%)

Total

No (%)

11-20 2(4) 1(2) 3(6)
21-30 1(2) 0(0) 1(2)
31-40 2(4) 4(8) 6(12)
41-50 9(18) 3(6) 12(24)
51-60 7(14) 6(12) 13(26)
61-70 10(20) 2(4) 12(24)
71-80 2(4) 1(2) 3(6)
Total 33(66) 17(34) 50(100)

 

Out of 50 focal liver lesions, 5 cases were unsatisfactory, 4 cases were cystic lesion, 2 cases were benign tumor and 39 cases were malignant tumor in cytology. Among the malignant cases, 12 were hepatocellular carcinoma (HCC) and 27 were metastatic adenocarcinoma (Figure 1).

 Figure 1. Pie diagram showing distribution of study cases according to FNA cytomorphology

Finally, 8 unsatisfactory, 3 benign and 33 malignant cases were diagnosed in cell block preparations. Among 33 malignant cases 6 were diagnosed as hepatocellular carcinoma (HCC) and 27 were diagnosed as metastatic adenocarcinoma (Figure-2).

 Figure 2. Pie diagram showing distribution of 44 cases according to combined cell block preparations.

 36 cases were conclusive on both cytomorphology and cell block preparations. On evaluation of cytomorphological diagnosis of 36 cases, 33 were true positive diagnosis, 2 were true negative diagnosis, 1 was false positive diagnosis and there was no false negative diagnosis (Table II). Sensitivity, specificity, PPV, NPV and accuracy of FNAC in the diagnosis of malignant focal liver lesions were100%, 66.67%, 97.06 %, 100 % & 97.22 %, respectively.

Table II: Statistical evaluation of cytomorphological diagnosis of 36 conclusive cases.

 

Combined cell block preparations (H&E and IHC) Cytomorphological diagnosis
Disease positive (Malignant) Disease negative(Benign)
Positive(Malignant)   33 TP               33 FP                  1
Negative(Benign)       3 FN                0 TN                  2
Total                         36                    33                        3

 

TP= True positive, TN= True negative, FP= False positive, FN= False negative

33 cases were diagnosed as malignant by both FNAC and cell block preparations. On evaluation of cytomorphological diagnosis, 4 were true positive, 23 were true negative, 4 were false positive and 2 were false negative in the detection of HCC (Table III). Sensitivity, specificity, PPV, NPV and accuracyof FNAC in the detection of HCC were 66.67%, 85.18%, 50%, 92% and 81.82%, respectively.

Table III: Statistical evaluation of cytomorphological diagnosis in the detection of HCC.

 

Combined cell block preparations (H&E and IHC) Cytomorphological diagnosis
Disease positive (HCC) Disease negative

(Non HCC)

Positive (HCC)                   6 TP               4 FP                   4
Negative (Non HCC)        27 FN               2 TN                  23
Total                                 33                     6                        27

 

TP= True positive, TN= True negative, FP= False positive, FN= False negative

Discussion

In the present study, USG guided FNAC was compared with cell block preparations (H&E and immunohistochemistry) in differentiation of focal liver lesions. FNA smears were available in all the 50 cases, but cell blocks were available in 44 cases.

Age of the study patients ranged from 15 to 80 years with a mean of 53.58 years. Nazir et al. (2010) and Kuo et al. (2004) showed 55 and 58.1 years as mean age in their studies which are close to the mean age of present study.7,8 Highest number of patients (26%) was in the age group of 51-60 years in our study. Nazir et al. (2010) reported that maximum number of cases was seen between 55-65 years of age which is nearly similar to present study.7 Out of 50 cases, 33 (66%) were male and 17 (34%) were female with male to female ratio of 1.94:1. Similar findings were reported by Swamy et al. (2011).9 Nazir et al. (2010) showed a male to female ratio of 1.7:1 which is also close to present study.7

Out of 50 cases, 5 (10%) cases were unsatisfactory, 4 (8%) cases were cystic lesion, 2 (4%) cases were benign tumor and 39 (78%) cases were malignant tumor on cytomorphology. Further categorization of benign tumors was not done as in Khurana et al. (2009).6 Among 39 malignant cases, 12 (30.77%) cases were HCC and 27 (69.23%) cases were metastatic carcinoma. All the cases of metastatic carcinoma were adenocarcinomas. Nearly similar findings were found on cytomorphology in the study of Mohmmed et al. (2012), Nazir et al. (2010), Khurana et al. (2009) and Ceyhan et al. (2006).6,7,10,11 Ozkara et al. (2012) found 9.9% of cases as unsatisfactory on cytomorphology which is similar to the unsatisfactory smear (10%) of the present study.12

In final diagnosis of 44 cases by combined cell block preparations (H&E and immunohistochemistry), 8 (18.18%) were unsatisfactory, 3 (6.82%) were benign tumors and 33 (75%) were malignant tumors. Nazir et al. (2010) reported 85% cases as malignant which is nearly close to the malignant cases found in the present study.7 But Mohmmed et al. (2012) showed 39% cases as malignant which is lower and Khurana et al. (2009) showed 93.75% cases as malignant which is higher than that of present study.6,10 Among the malignant lesions, 6 (18.18%) were HCC and 27 (81.82%) were metastatic adenocarcinoma in our study. Khurana et al. (2009) found 17.78% cases as HCC and 82.22% cases as metastatic tumor which are concordant with the present study.6

The sensitivity, specificity, and accuracy of USG guided FNAC in the evaluation of focal liver lesions were 100%, 66.67% and 97.22%, respectively. Sensitivity of the present study (100%) is similar or close to the sensitivity of studies done by Khurana et al. (2009), Nazir et al. (2010), Swamy et al. (2011) and Mohmmed et al. (2012).6,7,9,10 Specificity of the present study (66.67%) has concordance with the specificity found by Mohmmed et al. (2012).10 The specificity shown by Khurana et al. (2009), Nazir et al. (2010) and Swamy et al. (2011) has discordance with that of current study.6,7,9The present study showed an accuracy of 97.22% which is similar to that of Nazir et al. (2010) and Swamy et al. (2011).7,9

The sensitivity, specificity, and accuracy of FNAC in the detection of HCC were 66.67%, 85.18% and 81.82% respectively in our study.  Sensitivity of FNAC in the detection of HCC described by Ozkara et al. (2013) was 68.2% which is similar to the sensitivity of present study.12 Khurana et al. (2009) and Nazir et al. (2010) showed the sensitivity in the detection of HCC as 72.3% and 96% respectively which are higher than the sensitivity of present study.6,7 Specificity and accuracy showed by Nazir et al. (2010) were 100% and 97.5% respectively which are also higher than those of the present study.7

 Conclusion

FNAC of focal liver lesions has high sensitivity and accuracy in the detection of malignancy but it has low sensitivity in the detection of HCC. No significant complication was observed during aspiration. FNAC is a relatively safe, quick, cost effective and patient compliant procedure which has high accuracy in the differentiation between benign and malignant focal liver lesions. Simultaneous cell block preparations can improve the efficacy of FNAC in the subtyping of malignancy.

 References

  1. Marrero JA, Ahn J, Reddy KR. ACG clinical guideline: The Diagnosis and Management of Focal Liver Lesions. Am J Gastroenterol, 2014; 109(9): 1328-47.
  2. Conrad R, Prabhu SC, Cobb C, Raza A. Cytopathologic diagnosis of liver mass lesions. J Gastrointest Oncol, 2013; 4(1): 53-61.
  3. Nathan NA, Narayan E, Smith MM, Horn MJ. Cell block cytology: Improved Preparation and its Efficacy in Diagnostic Cytology. Am J Clin Pathol, 2000; 114: 599-606.
  4. Ali SR, Jayabackthan L, Rahim S, Sharel MB, Prasad K, Hegdekatte N. Role of fine needle aspiration cytology in the diagnosis of hepatic lesions. Muller J Med Sci Res, 2015; 6(2): 125-128.
  5. Ahuja A, Gupta N, Srinivasan R, Kalra N, Chawla Y, Rajwanshi A. Differentiation of Hepatocellular Carcinoma from Metastatic Carcinoma of the liver – Clinical and Cytological features. J Cytol, 2007; 24(3): 125-129.
  6. Khurana U, Handa U, Mohan H, Sachdev A. Evaluation of Aspiration Cytology of the Liver Space Occupying Lesions by Simultaneous Examination of Smears and Cell Blocks. Diagn Cytopathol, 2009; 37(8): 557-563.
  7. Nazir RT, Sharif MA, Iqbal M, Amin MS.Diagnostic Accuracy of Fine Needle Aspiration Cytology in Hepatic Tumours. J Coll Physicians Surg Pak, 2010; 20(6): 373-376.
  8. Kuo FY, Chen WJ, Lu SN, Wang JH, Eng HL. Fine Needle Aspiration Cytodiagnosis of Liver Tumors. Acta Cytologica, 2004; 48(2): 142-148.
  9. Swamy MCM, Arathi CA, Kodandaswamy CR. Value of ultrasonography-guided fine needle aspiration cytology in the investigative sequence of hepatic lesions with an emphasis on hepatocellular carcinoma. J Cytol, 2011; 28(4): 178-184.
  10. Mohmmed AA, Elsiddig S, Abdullhamid M, Gasim GI, Adam I. Ultrasound- guided fine needle aspiration cytology and cell block in the diagnosis of focal liver lesions at Khartoum Hospital, Sudan. Sudan JMS, 2012; 7(3): 183-187.
  11. Ceyhan K, Kupana SA, Bektas M et al. The diagnostic value of on-site cytopathological evaluation and cell block preparation in fine-needle aspiration cytology of liver masses. Cytopathol, 2006; 17: 267–274.
  12. Ӧzkara SK, Tuneli IӦ. Fine Needle Aspiration Cytopathology of Liver Masses: 101 cases with Cyto-/Histopathological Analysis. Acta Cytologica, 2013; 57:332-336

 

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.

Study of ER, PR and Ki-67 Expression in Different Histopathological Pattern of Endometrial Hyperplasia



Study of ER, PR and Ki-67 Expression in Different Histopathological Pattern of Endometrial Hyperplasia

*Fardousi F,1Sultana SS,2 Kaizer N,3 Dewan RK,4 Jinnah MS,5 Jeba R,6 Haque WS,7 Alam SM.8 Hussain M9

Abstract
Endometrial hyperplasia is one of the major gynaecological problem in peri and postmenopausal women worldwide. It deserves special attention because of its relationship with endometrial carcinoma. To study the histopathological pattern of endometrial hyperplasia in peri and postmenopausal women and their association with ER, PR and Ki-67 expression by immunohistochemistry is essential for early diagnosis with effective treatment. Out of 70 endometrial hyperplasia cases, 53 cases were simple endometrial hyperplasia without atypia, 8 cases were simple endometrial hyperplasia with atypia, 6 cases were complex endometrial hyperplasia without atypia and 3 cases were complex endometrial hyperplasia with atypia. ER expression in majority (35) of the cases of endometrial hyperplasia was between 51-100%. All the cases (3) of complex endometrial hyperplasia with atypia express ER less than 10%. The PR expression in majority of the cases (37) of endometrial hyperplasia was between 51-100%. All the cases (3) of complex endometrial hyperplasia with atypia expressed PR less than 40%. Ki-67 expression in majority of the cases of all types of endometrial hyperplasia 32 (78%) was less than 35%. All the cases (24) of simple endometrial hyperplasia without atypia had less than 35% Ki-67 expression. Only 6 cases had Ki-67 expression of more than 35%. In this study, 5 cases had negative (0%) Ki-67 expression. It can be inferred that after evaluating the ER, PR and Ki-67 expression, conservative treatment with progestogen and GnRH-agonists can be effective in selected cases of simple endometrial hyperplasia without or with atypia and complex endometrial hyperplasia without atypia.

[Journal of Histopathology and Cytopathology, 2018 Jul; 2 (2):74-84]

 Key words: Endometrial hyperplasia, Gynecological problem, Peri and postmenopausal women, Histopathological pattern, Immunohistochemistry, ER, PR and Ki-67 expression

 

  1. *Dr. Farzana Fardousi, Lecturer, Department of Cytopathology, National Institute of Cancer Research and Hospital (NICRH), Mohakhali, Dhaka. farzanafardousi@yahoo.com.
  2. Sk Salowa Sultana, Assistant Professor, Department of Pathology, Ad Din Women’s Medical College. Dhaka.
  3. Nahid Kaizer, Associate Professor (CC), Department of Pathology, Shahabuddin Medical College.
  4. Professor Dr. Rezaul Karim Dewan, Head of the Department of Pathology, Dhaka Medical College.
  5. Mohammed Shahed Ali Jinnah, Associate Professor, Department of Pathology, Dhaka Medical College.
  6. Ruksana Jeba, Associate Professor, Department of Pathology, Dhaka Medical College.
  7. Lt Col (Dr) Wasim Selimul Haque. Classified spl in Pathology, Armed Forces Institute of Pathology.
  8. Prof Brig Gen Dr. SM. Mahbubul Alam, Ex Deputy Commandment, Armed Forces Institute of Pathology, Senior consultant- Histopathology, Apollo Hospitals Dhaka.
  9. Professor Dr. Maleeha Hussain, Ex Head of the Department of Pathology, Dhaka Medical College.

 

*For corespondence

 Introduction
Endometrial hyperplasia has a high risk for malignant transformation into endometrial carcinoma in peri and postmenopausal women.1,2 Eendometrial hyperplasia usually develops due to the continuous estrogen stimulation unopposed by progesterone. In the years before menopause, women may have numerous cycles without ovulation (anovulatory) during which there is sustained and unopposed estrogen activity. It is also likely that hormone replacement therapy (HRT) consisting of estrogen without progesterone may lead to endometrial hyperplasia.3 The postmenopausal endometrium which  despite being atrophic, retain a weak proliferative pattern for many years probably as a response to continuous low level of estrogenic stimulation. These are at a higher risk of progression to endometrial hyperplasia and subsequently to endometrial malignancy. Although the diagnosis of endometrial hyperplasia can be made by histopathological examination; the immunohistochemistry aids for the prognosis, in order to establish the best treatment.4

The receptor status for estrogen and progesterone in endometrial hyperplasia can be a prognostic indicator in the treatment. The expression of estrogen and progesterone receptors varies in different types of endometrial hyperplasia. Several studies have found that, all types of hyperplasia present a smaller number of receptors compared to the endometrium in proliferative phase, but higher compared with secretory endometrium and endometrial carcinoma.5 The proliferative activity of endometrial hyperplasia can be examined by using an antibody to Ki-67 antigen, a non-histone protein, a well established marker of proliferative activity. The expression of the human Ki-67 protein is strictly associated with cell proliferation.

The endometrium of reproductive-aged women undergoes cyclic developmental changes in response to the steroids – estrogen and progesterone. The highest score of estrogen and progesterone receptors are observed in the epithelial and stromal cells of the normal uterine endometrium at the early proliferative phase; then, throughout the secretory phase, the ER and PR scores decline. Again, the highest score of estrogen receptors and progesterone receptors are observed in non-atypical hyperplasia and lowest score of estrogen receptors and progesterone receptors are observed in atypical hyperplasia.6

Ferrandina et al. (2005)7 found that, ER and PR positive cases of endometrial hyperplasias had a statistically significant association with the clinicopathological parameters, which correlates with a more favorable prognosis. In addition, the hormone receptor status appears to correlate with the treatment response to the progesterone therapy. This finding may be of particular clinical importance, since almost all endometrial hyperplasia cases contain estrogen/ progesterone receptors and progesterone therapy could be beneficial in this cases.8

The expression of the human Ki-67 protein is strictly associated with cell proliferation. During interphase, the antigen can be exclusively detected within the nucleus, whereas in mitosis most of the protein is relocated to the surface of the chromosomes. The fact that the Ki-67 protein is present during all active phases of the cell cycle (G(1), S, G(2), and mitosis), but is absent from resting cells G(0), makes it an excellent marker for determining the so-called growth fraction of a given cell population. The highest score of Ki-67 expression is observed in atypical hyperplasia and lowest score of Ki-67 expression is observed in non-atypical hyperplasia. Scholzen and Gerdes, (2009)9 in their study showed that, cell proliferation is highly correlated with Ki67 expression.

Perez-Medina et al have observed that, ER, PR and Ki-67 expression rate, along with the histopathological features of endometrial hyperplasia are important prognostic factors.10 After evaluating the ER, PR and Ki-67 expression rate, they have shown that, for all cases of endometrial hyperplasia, the general treatment protocol i.e. hysterectomy is not rationally justified. Patients who want to complete their family or who have heart disease or surgical or anaesthetic risk history, if they have positive ER, PR expression and low Ki-67 expression; they can be selected for conservative treatment with progestogens and GnRH-agonists. These patients should be followed for 5 years by biopsies every 6 months.

Hysterectomy is usually recommended for cases with endometrial hyperplasia.2 Several studies have revealed that less than 5 % of simple and complex non-atypical hyperplasia cases undergo malignant transformation in the long term (20 years), whereas this percentage increases to 30 % when there is an atypical hyperplasia.11 So, the rationality of hysterectomy in the treatment of endometrial hyperplasia in general needs to be evaluated.

The evaluation of receptor status by markers for hormone receptors (estrogen and progesterone receptors) and proliferative activity by proliferative marker (Ki-67) in patients with endometrial hyperplasia can predict the treatment option in selected cases.12 -16

 Methods
This is a descriptive cross sectional study which was carried out at the Department of Pathology, Dhaka Medical College, and Dhaka during the period of January 2013 to December 2014. A total of 110 peri and postmenopausal women with dysfunctional uterine bleeding or postmenopausal bleeding who underwent D&C or hysterectomy were screened.  A total of seventy histopathologically diagnosed cases of endometrial hyperplasia who met the enrollment criteria (inclusion & exclusion criteria) were included in this study. Among 70 cases, endometrial curettage biopsy specimens were 45 and hysterectomy specimens were 25. Routine Hematoxylin and Eosin staining was done on all 70 samples. Out of these 70 cases, ER, PR and Ki-67 immunostaining was done on 41 cases. Ethical clearance was taken for this study from institutional ethical committee of Dhaka Medical College. Each patient was interviewed before collection of the specimen and relevant information was recorded in a prescribed clinical proforma. Detail history with particular attention to age, clinical features, age at menarche, parity, obesity, history of contraceptives, history of hormone replacement therapy, history of diabetes, history of hypertension, history of estrogen producing ovarian tumor, age at menopause were taken.

 Histopathological examination

The gross examination of specimens, routine tissue processing and Hematoxylin & Eosin staining were done at the Department of Pathology, Dhaka Medical College.

 Microscopic analysis

According to WHO classification endometrial hyperplasia is classified into simple endometrial hyperplasia without atypia, Simple endometrial hyperplasia with atypia, Complex endometrial hyperplasia without atypia, Complex endometrial hyperplasia with atypia.17

 Immunohistochemical examination

Immunostaining for ER, PR and Ki-67 was done at AFIP (Armed Forces Institute of Pathology, Dhaka). A total of 41 cases were selected for immunohistochemical examination for ER, PR and Ki-67 expression. For immunostain, 3cases of proliferative phase of endometrium, 3 cases of secretory phase of endometrium and 3 cases of well differentiated endometrial carcinoma were enrolled as control.

All the data were recorded in data sheet along with patient’s clinical findings. The main objectives of this study were to observe the histopathological pattern of endometrial hyperplasia in peri and postmenopausal women and their association with ER, PR and Ki-67 expression by immunohistochemical method.

1) Primary antibody –Mouse monoclonal Anti-Human Estrogen Receptor, clone 1D5, code M7047(1:60 dilution), Mouse monoclonal Anti-Human Progesterone Receptor, clone PgR636(1:50 dilution) and Mouse monoclonal antibody against ki67 antigen(1:100 dilution)  were used as primary antibody.

2) Secondary antibody – Envision (ready to use, Dako), was used as secondary antibody.

3) Positive control –

  1. High grade breast carcinoma was taken as positive control for ER and PR.
  2. Follicular hyperplasia of lymph node was taken as positive control for Ki-67.

 Immunohistochemical analysis

For ER and PR expression in endometrial tissue: 18

Score for proportion staining Score for staining intensity
1. 0-25 % nuclei 1. Absent or weak staining
2. 26-75 % nuclei 2. Strong staining
3. ≥ 76 % nuclei 3. Very strong staining

 

Cat – I Total score 2 Immuno negative
Cat – II Total score 3-4 Immuno reactive
Cat – III Total score 5-6 Immuno reactive

For Ki-67 expression in endometrial tissue:

The patients are divided into two groups:

  1. Low Ki-67 expression (≤ 35 % cells are Ki-67 positive)
  2. High Ki-67 expression (> 35 % cells are Ki-67 positive)

(Nuclear staining in endometrial glandular epithelial cells are evaluated)

 Statistical analysis

Statistical analyses of the results were obtained by using window based computer software devised with Statistical Packages for Social Sciences (SPSS-16). Percentages were calculated to find out the proportion of the findings. The results are presented in Tables and Figures.

 Results
A total of 70 histopathologically diagnosed endometrial hyperplasia cases were included in this study.  Among 70 cases endometrial curettage biopsy specimens were 45 and hysterectomy specimens were 25. Out of all endometrial hyperplasia cases, 53 cases were simple endometrial hyperplasia without atypia, 8 cases were simple endometrial hyperplasia with atypia, 6 cases were complex endometrial hyperplasia without atypia and 3 cases were complex endometrial hyperplasia with atypia. Out of these all the 8 cases of simple endometrial hyperplasia with atypia, 6 cases of complex endometrial hyperplasia without atypia, 3 cases of complex endometrial hyperplasia with atypia and 24 cases of simple endometrial hyperplasia without atypia.

Table I: Distribution of the study patients by immunohistochemistry findings (ER %) (n=50)

 

Immunohisto-chemistry
findings (ER %)
PP

(n=3)

SP

(n=3)

SEH without atypia

(n=24)

SEH with atypia

(n=8)

CEH without atypia

(n=6)

CEH with atypia

(n=3)

End. Ca. grade 1

(n=3)

Total

(n=50)

n %
≤10 0 1 0 0 0 3 1 5 10.0
11-20 0 0 0 0 0 0 1 1 2.0
21-30 0 0 0 0 0 0 1 1 2.0
31-40 0 1 0 0 0 0 0 1 2.0
41-50 0 0 3 0 0 0 0 3 6.0
51-60 0 0 2 2 0 0 0 4 8.0
61-70 0 1 2 0 1 0 0 4 8.0
71-80 0 0 10 0 0 0 0 10 20.0
81-90 2 0 5 5 4 0 0 16 32.0
91-100 1 0 2 1 1 0 0 5 10.0
Total 3 3 24 8 6 3 3 50 100

(PP- Proliferative phase of endometrium, SP- Secretory phase of endometrium, SEH without atypia- Simple endometrial hyperplasia without atypia, SEH with atypia- Simple endometrial hyperplasia with atypia, CEH without atypia – Complex endometrial hyperplasia without atypia, CEH with atypia- Complex endometrial hyperplasia with atypia and End. Ca. grade 1- Endometrial carcinoma Grade-I)

 Distributions of the patients with PR expression (%). PR expression in majority of the cases (37) of endometrial hyperplasia is between 51-100%. All the cases (3) of complex endometrial hyperplasia with atypia express PR less than 40% (Table II).

 Table II: Distribution of the study patients by immunohistochemistry findings (PR %) (n=50)

 

Immunohist-ochemistry
findings (PR %)
PP

(n=3)

SP

(n=3)

SEH without atypia

(n=24)

SEH with atypia

(n=8)

CEH without atypia

(n=6)

CEH with atypia

(n=3)

End. Ca. grade 1

(n=3)

Total

(n=50)

n %
≤10 1 0 0 0 0 0 0 1 2.0
11-20 0 0 0 0 0 2 1 3 6.0
21-30 0 0 0 0 0 0 0 0 0.0
31-40 0 0 1 0 0 1 0 2 4.0
41-50 0 0 0 0 0 0 0 0 0.0
51-60 0 0 3 0 0 0 0 3 6.0
61-70 0 0 2 0 0 0 1 3 6.0
71-80 0 0 6 0 0 0 0 6 12.0
81-90 2.0 1 6 5 1 0 1 16 32.0
91-100 0 2 6 3 5 0 0 16 32.0
Total 3 3 24 8 6 3 3 50 100

(PP- Proliferative phase of endometrium, SP- Secretory phase of endometrium, SEH without atypia- Simple endometrial hyperplasia without atypia, SEH with atypia- Simple endometrial hyperplasia with atypia, CEH without atypia – Complex endometrial hyperplasia without atypia, CEH with atypia- Complex endometrial hyperplasia with atypia and End. Ca. grade 1- Endometrial carcinoma Grade-I)

Table-III shows distributions of the patients with Ki-67 expression (%). Ki-67 expression in majority of the cases of all types of endometrial hyperplasia 32(78%) is less than 35%.

 Table III: Distribution of the study patients by immunohistochemistry findings (Ki-67%) (n=50)

 

Immunohisto
-chemistry
findings (KI-67%)
PP

(n=3)

SP

(n=3)

SEH without atypia

(n=24)

SEH with atypia

(n=8)

CEH without atypia

(n=6)

CEH with atypia

(n=3)

End. Ca. grade 1

(n=3)

Total

(n=50)

n %
0 0 0 4 0 1 0 0 5 10.0
1-10 0 2 10 1 1 0 0 14 28.0
11-20 0 1 1 2 2 0 0 6 12.0
21-30 0 0 3 0 1 0 0 4 8.0
31-35 0 0 6 3 0 0 1 12 24.0
36-50 0 0 0 0 1 2 0 1 2.0
51-60 1 0 0 2 0 1 1 5 10.0
61-70 0 0 0 0 0 0 0 0 0.0
71-80 1 0 0 0 0 0 0 1 2.0
81-90 1 0 0 0 0 0 0 1 2.0
91-100 0 0 0 0 0 0 1 1 2.0
Total 3 3 24 8 6 3 3 50 100

(PP- Proliferative phase of endometrium, SP- Secretory phase of endometrium, SEH without atypia- Simple endometrial hyperplasia without atypia, SEH with atypia- Simple endometrial hyperplasia with atypia, CEH without atypia – Complex endometrial hyperplasia without atypia, CEH with atypia- Complex endometrial hyperplasia with atypia and End. Ca. grade 1- Endometrial carcinoma Grade-I)

(SEH without atypia- Simple endometrial hyperplasia without atypia, SEH with atypia- Simple endometrial hyperplasia with atypia, CEH without atypia – Complex endometrial hyperplasia without atypia, CEH with atypia- Complex endometrial hyperplasia with atypia I)

 

Figure 1.  Pie chart showing distribution of the patients by diagnosis (n=70)

Pie chart showing the commonest diagnosis in 70 patients was SEH without atypia (75.7%) followed by SEH with atypia (11.4%) (Fig 1).

Discussion
Endometrial hyperplasia has a significant place in gynecological morbidity in women of reproductive age (10% to 18%).19 Endometrial hyperplasia is associated with menstrual irregularities and anaemia in women and poses a high risk for malignant transformation into endometrial cancer.20 World wide endometrial cancer is the most common gynecological cancer in peri and postmenopausal women.21,22 The incidence of endometrial adenocarcinoma not only has remained high but in recent years has tended to significantly increase in many countries, including Bangladesh.19,23-32

In this study, the commonest diagnosed lesion was simple endometrial hyperplasia without atypia which was 53(75.7%) followed by simple endometrial hyperplasia with atypia 8(11.4%), complex endometrial hyperplasia without atypia 6(8.6%) and then complex endometrial hyperplasia with atypia 3(4.3%).

From the present study it was observed that, in all types of endometrial hyperplasia except complex endometrial hyperplasia with atypia, the ER expression was lower than proliferative phase but higher than secretory phase of endometrium. However, the ER expression in complex endometrial hyperplasia with atypia was lower than any other type of endometrial hyperplasia or proliferative phase or secretory phase of endometrium. It was even lower than endometrial carcinoma. Similar study done by Ilie et al. (2011)33 also found that the ER expression in different types of endometrial hyperplasia and endometrial cancer is lower than proliferative phase but higher than secretory phase of endometrium.33 Found that, the expression of ER in different types of endometrial hyperplasia was much lower (41.5%) than the present study (75%) except that of complex endometrial hyperplasia with atypia. This variation may be due to inclusion of menopausal patients in their study.

In this study it was observed that, the mean ER expression was 75±15.1% in simple endometrial hyperplasia without atypia. Similar observations was found by Goncharenko et al. (2013)20 they have found 75.6% ER expression in simple endometrial hyperplasia without atypia. This may be due to inclusion of perimenopausal women in their study like present study. It was also observed that the mean ER expression was 10±0% in complex endometrial hyperplasia with atypia in the current study. These findings differed from Goncharenko et al. (2013)20 they found 65.2% ER expression in complex endometrial hyperplasia with atypia. This variation may be due to inclusion of many patients in their study at their reproductive age.

From the present study it was observed that, the  PR expression  in all types of endometrial hyperplasia except complex endometrial hyperplasia with atypia, was higher than proliferative phase of normal endometrium and endometrial carcinoma ,but they were lower than secretory phase of normal endometrium. However, the PR expression was lowest in complex endometrial hyperplasia with atypia.  In the study done by Ilie et al. (2011)33 found that, the PR expression in different types of endometrial hyperplasia was lower than proliferative phase but higher than secretory phase of endometrium and endometrial carcinoma. This variation may be due to inclusion of only menopausal women in their study. In this current study it was observed that, the mean PR expression was 80.8±16.4% in simple endometrial hyperplasia without atypia. This finding was nearly similar to the observations found by Goncharenko et al. (2013).20 They have found 69.3% PR expression in simple endometrial hyperplasia without atypia as they also included perimenopausal patients in their study.

It was observed in our study that the mean PR expression was 25±8.7% in complex endometrial hyperplasia with atypia. These findings differed from Goncharenko et al. (2013)20 they found 44.3% PR expression in complex endometrial hyperplasia with atypia. This may be due to inclusion of many patients in their study at their reproductive age. This result is in concordance with that of, Uchikawa  et al. (2003)5 in which it was found that, the expression of ER and PR decreased in endometrial hyperplasia compared with normal proliferative endometrium.

In present study, the expression of Ki-67 was compared with proliferative phase (73.3±12.6%) and secretory phase (12.3±5.8 %) of normal endometrium and with endometrial carcinoma, grade I (66.7±30.5%). We found that, the presence of mitotic activity in normal endometrium was observed more in the proliferative phase than the secretory phase. Mitotic activity in neoplastic and hyperplasic endometrium was low compared with proliferative phase of normal endometrium, but more than the endometrium in secretory phase.This findings are similar to the study done by Ilie et al. (2011).33

In the present study, the mean Ki-67 expression was 36.3±18.5% in simple endometrial hyperplasia with atypia. This finding are nearly similar to the study done by Goncharenko et al. (2013)20 who found upto 50% Ki-67 expression in simple endometrial hyperplasia with atypia. This may be due to inclusion of perimenopausal patients like our study. The present study result is in concordance with that of Uchikawa et al. (2003)5 in which they found that, ki-67 expression was more in atypical hyperplasia (50%) than non-atypical hyperplasia (10%).

In patients with simple endometrial hyperplasia with atypia and complex endometrial hyperplasia without atypia, Ki-67 expression can predict the treatment protocol. The patients with low Ki-67 expression can be selected for conservative treatment with hormone therapy and with high Ki-67 expression should undergo hysterectomy.  In complex endometrial hyperplasia with atypia, they should preferably be treated by hysterectomy operation. If, patients who want to complete their family and have low Ki-67 expression, they can be temporarily treated by conservative treatment. After delivery, they must undergo surgical intervention.16

The treatment response with hormone therapy depends on expression of ER & PR. So, before hormonal intervention, ER & PR should be evaluated, because in few cases, where expressions are low, have the chance of poor response to treatment. 10

 Limitation of the study

The study population was selected from the department of Pathology of Dhaka Medical College Hospital. But there are many patients with endometrial hyperplasia who are attending other hospitals than DMCH. Therefore, the sample lacks representation of the population. Thus, the study place was selected purposively and the respondents, those are interviewed, were attended a particular department of a specific hospital.

 Conclusion

The study revealed that highest ER & PR expression were observed in complex endometrial hyperplasia without atypia and lowest ER & PR expression were observed in complex endometrial hyperplasia with atypia. Again, highest Ki-67 expression was observed in complex endometrial hyperplasia with atypia and lowest Ki-67 expression was observed in simple endometrial hyperplasia without atypia. In complex endometrial hyperplasia with atypia, if patient desires to complete the family, after evaluation of ER, PR and Ki-67 expression, conservative treatment can be given and allowed to conceive. After delivery, if Ki-67 expression remains high, then hysterectomy must be done.

 Recommendations

A large follow–up study is recommended for patients of endometrial hyperplasia selected for conservative treatment with progestogen and GnRH-agonists. Monitoring should be done by observing the Ki-67 expression in these patients. If the Ki-67 expression increases, they should be treated by surgical intervention.

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